Mipol1-etv1 gene rearrangements

ABSTRACT

Compositions and methods associated with recurrent MIPOL1-ETV1 genetic rearrangements that are useful for cancer diagnosis and therapy are disclosed.

This application is a continuation of U.S. patent application Ser. No.15/084,721, filed Mar. 30, 2016, issued as U.S. Pat. No. 9,719,143,which is a continuation of U.S. patent application Ser. No. 12/667,819,filed Oct. 18, 2010, issued as U.S. Pat. No. 9,303,291, which is anational phase application under 35 U.S.C. §371 of InternationalApplication No. PCT/US2008/069201, filed Jul. 3, 2008, which claimspriority to U.S. Provisional Application 60/958,629, filed Jul. 6, 2007,each of which are herein incorporated by reference in their entireties.

This invention was made with government support under CA069568 andCA111275 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to compositions and methods for cancer diagnosis,research and therapy, including but not limited to, cancer markers. Inparticular, this invention relates to recurrent MIPOL1-ETV1 geneticrearrangements that are useful as diagnostic markers and clinicaltargets for prostate cancer.

BACKGROUND OF THE INVENTION

Cancer research may identify altered genes that are causally implicatedin oncogenesis. Several types of somatic mutations that result inaltered activity of an oncogene or tumor suppressor gene have beenidentified, including base substitutions, insertions, deletions,translocations, and chromosomal gains and losses. Compelling evidenceexists for a causal role for some chromosomal rearrangements in cancer(Rowley, Nat Rev Cancer 1: 245 (2001)). Recurrent chromosomalaberrations have been primarily characteristic of leukemias, lymphomas,and sarcomas. Less than 1% of the known, disease-specific chromosomalrearrangements are associated with epithelial tumors (carcinomas),although those cancers are much more common and contribute to arelatively large fraction of the morbidity and mortality associated withhuman cancer (Mitelman, Mutat Res 462: 247 (2000)). While hematologicalmalignancies are often characterized by disease-specific chromosomalrearrangements, most solid tumors have a plethora of non-specificchromosomal aberrations. Karyotypic complexity of solid tumors isthought to result from secondary alterations acquired through cancerevolution or progression.

Cancer-related chromosomal rearrangements may result from two primarymechanisms. In one, promoter/enhancer elements of one gene arerearranged adjacent to a proto-oncogene, thus causing altered expressionof an oncogenic protein. This type of translocation is exemplified bythe apposition of immunoglobulin (IG) and T-cell receptor (TCR) genes tothe MYC oncogene, leading to oncogene activation in B- and T-cellmalignancies, respectively (Rabbitts, Nature 372: 143 (1994)). In theother mechanism, rearrangement results in the fusion of two genes, whichproduces a fusion protein that may have a new function or alteredactivity. This type of translocation is exemplified by the BCR-ABL genefusion in chronic myelogenous leukemia (CML) (Rowley, Nature 243: 290(1973); de Klein et al., Nature 300: 765 (1982)), which led to therational development of imatinib mesylate that successfully targets theBCR-ABL kinase (Deininger et al., Blood 105: 2640 (2005)).

Recurrent MIPOL1-ETV1 genetic rearrangements are described herein, whichare useful for diagnosis and therapeutic applications related to humanepithelial tumors.

SUMMARY OF THE INVENTION

A method is disclosed for diagnosing prostate cancer comprisingdetecting the presence or absence in a biological sample of aMIPOL1-ETV1 genetic rearrangement, wherein the presence in the sample ofthe genetic rearrangement is indicative of prostate cancer in theindividual from whom the sample was obtained. In some embodiments, thesample is tissue, blood, plasma, serum, urine, semen, prostaticsecretions or prostate cells. In some embodiments, the detecting stepcomprises detecting chromosomal rearrangements of genomic DNA encodingMIPOL1 and ETV1. The detecting step may use a nucleic acid sequencingtechnique or a nucleic acid hybridization technique, such as in situhybridization (ISH), hybridization to one or more moieties in amicroarray, or Southern blot analysis. In some embodiments, thedetecting step further includes nucleic acid amplification, which mayuse known methods that include, but are not limited to, polymerase chainreaction (PCR), reverse transcription polymerase chain reaction(RT-PCR), transcription-mediated amplification (TMA), ligase chainreaction (LCR), strand displacement amplification (SDA), and nucleicacid sequence based amplification (NASBA). In some embodiments, thedetecting step detects mRNA associated with MIPOL1-ETV1 geneticrearrangements or protein expression resulting from MIPOL1-ETV1 geneticrearrangements. Such detection may include analysis of RNA and/orprotein expression levels, or determination of sequence characteristics.

Compositions are disclosed for diagnosing prostate cancer comprising areagent that directly or indirectly detects a junction between ETV1genetic material and MIPOL1 genetic material associated with aMIPOL1-ETV1 genetic rearrangement. Embodiments of such reagents include:a probe comprising a sequence that hybridizes to the junction betweenETV1 genetic material and MIPOL1 genetic material associated with aMIPOL1-ETV1 genetic rearrangement, which may be the junction at which aETV1 gene is inserted into a MIPOL1 gene; a combination of first andsecond probes, in which a first probe comprises a sequence thathybridizes to the ETV1 gene and a second probe comprises a sequence thathybridizes to the MIPOL1 gene; and at least one first amplificationoligonucleotide that comprises a sequence that hybridizes specificallyto a ETV1 gene and at least one second amplification oligonucleotidethat comprises a sequence that hybridizes specifically to an MIPOL1gene.

In some embodiments, the composition of amplification oligonucleotidesmay also include a probe that hybridizes specifically to a sequencelocated between the sequences hybridized by the first amplificationoligonucleotide and the second oligonucleotide, which probe mayhybridize specifically to a sequence in the ETV1 gene or in the MIPOL1gene. All probes may be linked directly or indirectly to a label thatprovides a detectable signal.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the entire ETV1 locus rearranged to 14q13.3-14q21.1in LNCaP and MDA-PCa 2B prostate cancer cells. FIG. 1, a is a schematicillustration of the ETV1 locus on chromosome 7 and FIG. 1, b is aschematic illustration of 14q13.3-14q21.1 on chromosome 14 and BACs usedas probes for fluorescence in situ hybridization (FISH) to detectgenetic rearrangements. FIG. 1, c and -d illustrate FISH performed byusing BACs indicated with the corresponding fluorescent label onmetaphase spreads from LNCaP (tetraploid) cells (FIG. 1, c) and d)MDA-PCa 2B (diploid) cells (FIG. 1, d) to detect rearrangements at theETV1 locus (left panel) and 14q13.3-14q21.1 (right panel). FIG. 1, e-g,illustrate structure of ETV1 and 14q13.3-14q21.1 in: normal cells (FIG.1, e), LNCaP cells (FIG. 1, f), and MDA-PCa 2B cells (FIG. 1, g), asdetermined by FISH for all.

FIG. 2 shows that the ETV1 locus is rearranged to chromosome 14 in LNCaPcells. FIG. 2, a-b, are schematic illustrations of BACs used fromchromosomes 7p and 14q32, respectively (previously FISH mapped tochromosome 14). FIG. 2, c. illustrates FISH using BACs labeled with theindicated fluorescent label showed two copies of ETV1 on chromosome 7and two copies on chromosome 14, as identified by RP11-483K13.

FIG. 3 shows the identification of the genomic breakpoint at the ETV1locus in LNCaP cells. FIG. 3, a illustrates FISH used to narrow thebreakpoint region between BACs 12 and 2, as shown in green in FIG. 3, b,as BACs 1 and 12 co-localized to chromosome 7 and chromosome 14. FIG. 3,b illustrates a series of 22 probes (A-Q) for Southern blotting thatwere designed to span the implicated region. FIG. 3, c illustrates arestriction map of the Probe A region, with restriction sites for PstIand EcoRI indicated, which was the only probe to show rearranged bandson Southern blotting. FIG. 3, d illustrates Southern blotting with ProbeA which showed additional bands in LNCaP genomic DNA digested with EcoRIand PstI, but not in VCaP, normal male (“Nor male”) genomic DNA or humanplacental DNA.

FIG. 4 shows inverse PCR that identifies the insertion of ETV1 into theMIPOL1 locus on chromosome 14. FIG. 4, a illustrates, a series ofdivergent PCR primers designed for inverse PCR (A3-B3) which were basedon the detection of rearrangements in LNCaP genomic DNA digested withEcoRI and PstI using Probe A. FIG. 4, b illustrates results obtainedfrom PCR amplification using nested PCRs subsequently with A1 and B1(1), A2 and B2 (2), and A3 and B3 (3) on PstI digested low (L) and high(H) passage LNCaP genomic DNA. FIG. 4, c illustrates the sequence of thenested PCR product of A3 and B3 (shown in FIG. 4, b), in which the DNAbreakpoint is shown in red, and the partner sequence was intronic DNAfrom the MIPOL1 locus at 14q13.3-14q21.1. FIG. 4, d illustrates thebreakpoint location shown by an asterisk. FIG. 4, e illustrates primersdesigned (as shown in FIG. 4, c) to confirm the fusion by PCR onundigested DNA, in which PCR confirmed the fusion in both low and highpassage LNCaP cells, but not VcaP, normal male (“Nor male”) genomic DNAor human placental DNA.

FIG. 5 shows that the 7p telomere is retained on all copies ofchromosome 7 in LNCaP, by using FISH performed using chromosome 7centromeric and 7p telomere probes on LNCaP metaphases. All four copiesof chromosome 7 identified by the centromeric probes retained their 7ptelomeric sequence, demonstrating that the ETV1 rearrangement is not atranslocation nor involves a telomeric deletion at 7p.

FIG. 6 shows that ETV1 and the contiguous genes at 14q13.3-14q21.1 arecoordinately regulated in prostate cancer, LNCaP and the androgeninsensitive LNCaP derivative C4-2B. FIG. 6, a illustrates the tissuespecificity of the 4 contiguous genes in the 14q13.3-14q21.1 regionwhich was interrogated in the expo dataset (using Oncomine) andexpression (in normalized expression units) is shown for 28 distincttumor types and prostate cancer. FIG. 6, b and -c illustrate androgenregulation of the contiguous 14q13.3-14q21.1 genes (FIG. 6, b) and ETV1(FIG. 6, c), as determined by qPCR in LNCaP cells with (+) or without(−) stimulation by the synthetic androgen R1881. Ratios of target geneto GAPDH (mean (n=4)+S.E.) are shown. FIG. 6, d illustrates that LNCaPand its androgen insensitive derivative, C4-2B, were profiled using amicroarray (Agilent Whole Genome Microarrays), in which the top ten mostdifferentially expressed features (LNCaP/C4-2B) are shown. FIG. 6, e-gillustrate qPCR results that show decreased expression of androgenregulated genes in C4-2B compared to LNCaP, for markers ETV1 (FIG. 6, e)and PSA (FIG. 6, f), and the 4 contiguous transcripts at 14q13.3-14q21.1(FIG. 6, g).

FIG. 7 shows that MDA-PCa 2B has outlier expression of ETV1, in whichexpression of ERG and ETV1 was determined by qPCR for the prostatecancer cell lines MDA-PCa 2B, LNCaP, PC3, NCI-H660, 22RV1, VCaP, DU145,LAPC4 and the immortalized benign prostate epithelial cell line RWPE,and the amounts of ERG and ETV1 for each sample were normalized to theaverage amount of GAPDH and HMBS for each sample.

FIG. 8 shows that the LNCaP androgen insensitive derivative C4-2Bharbors the same ETV1 rearrangement as the parental LNCaP cell line.FIG. 8, a is a schematic of the ETV1 locus on chromosome 7 and BACs usedas probes for fluorescence in situ hybridization (FISH) to detectrearrangements at the ETV1 locus, in which the breakpoint locationsidentified in LNCaP are indicated by an asterisk. FIG. 8, b illustratesresults obtained by FISH performed by using BACs indicated with thecorresponding fluorescent label on C4-2B (tetraploid). FIG. 8, cillustrates results obtained by PCR using primers from chromosomes 7 and14 which confirmed the rearrangement of the ETV1 locus in C4-2B,similarly to low and high passage LNCaP, and in which no products wereobtained from other prostate cancer cell lines, human placental DNA, ornormal male human DNA.

DEFINITIONS

To facilitate an understanding of this disclosure, terms are definedbelow:

As used herein, “gene rearrangement” or “genetic rearrangement” refersto any altered arrangement of genomic DNA resulting from the chromosomalrearrangement of two distinct genetic regions, two different genes, orportions of two different genes. The product of a genetic rearrangementincludes a fusion of genetic material that does not exist in the absenceof the genetic rearrangement. That is, the genetic rearrangement resultsin a junction that fuses one genetic sequence to another geneticsequence to produce a junction does not exist in a wild typenon-rearranged genome. Examples of genetic rearrangements include, butare not limited to, insertions of all or part of a first gene or geneticlocus into a second gene or genetic locus. In some cases, an insertionmay result in a new configuration of the first and second genes orgenetic loci that changes regulation of expression, e.g., resulting inincreased or decreased expression of a wild type protein. In othercases, an insertion may result in a gene fusion or chimeric DNA that istranscribed to make a chimeric RNA, which may be translated to makealtered protein(s) compared to wild type protein(s), i.e., resulting ina new product that is different than made in a wild type configuration.

As used herein, “gene fusion” may refer to a chimeric genomic DNA, achimeric mRNA that is transcribed from a chimeric DNA (which may bereferred to as “gene fusion RNA”), or a protein translated from chimericmRNA which is transcribed from a chimeric DNA. Such a “gene fusionprotein” (or “gene fusion polypeptide”) may be a truncated proteincompared to wild type protein, or a chimeric protein resulting fromexpression of at least a portion of a first gene fused to at least aportion of a second gene. A gene fusion (or RNA transcribed from thegene fusion DNA) need not include entire genes or exons of genes.

As used herein, the term “transcriptional regulatory region” refers tothe non-coding upstream regulatory sequence of a gene, also called the5′ untranslated region (5′UTR).

As used herein, the terms “detect”, “detecting”, or “detection” maydescribe either the general act of discovering or discerning or thespecific observation of a detectably labeled composition.

As used herein, the term “inhibits at least one biological activity of agene fusion” refers to any agent that decreases any activity of a genefusion disclosed herein, its transcript(s) and/or translation products(e.g., including, but not limited to, the activities described herein),via directly contacting a gene fusion protein, contacting gene fusionmRNA or genomic DNA, causing conformational changes of gene fusionpolypeptides, decreasing gene fusion protein levels, or interfering withgene fusion interactions with signaling partners, and affecting theexpression of target gene fusions (i.e., transcription or translation ofthe resulting transcripts). Inhibitors also include molecules thatindirectly regulate gene fusion biological activity by interceptingupstream signaling molecules.

As used herein, the term “siRNAs” refers to small interfering RNAs. Insome embodiments, siRNAs comprise a duplex, or double-stranded region,of about 18-25 nucleotides long; often siRNAs contain from about two tofour unpaired nucleotides at the 3′ end of each strand. At least onestrand of the duplex or double-stranded region of a siRNA issubstantially homologous to, or substantially complementary to, a targetRNA molecule. The strand complementary to a target RNA molecule is the“antisense strand;” the strand homologous to the target RNA molecule isthe “sense strand,” and is also complementary to the siRNA antisensestrand. siRNAs may also contain additional sequences; non-limitingexamples of such sequences include linking sequences, or loops, as wellas stem and other folded structures. siRNAs appear to function as keyintermediaries in triggering RNA interference in invertebrates and invertebrates, and in triggering sequence-specific RNA degradation duringposttranscriptional gene silencing in plants.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression by siRNAs. It is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi may also be considered to inhibit the function of a target RNA; thefunction of the target RNA may be complete or partial.

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor and the extent of metastases (e.g.,localized or distant).

As used herein, the term “gene transfer system” refers to any means ofdelivering a composition comprising a nucleic acid sequence to a cell ortissue. For example, gene transfer systems include, but are not limitedto, vectors (e.g., retroviral, adenoviral, adeno-associated viral, andother nucleic acid-based delivery systems), microinjection of nakednucleic acid, polymer-based delivery systems (e.g., liposome-based andmetallic particle-based systems), biolistic injection, and the like. Asused herein, the term “viral gene transfer system” refers to genetransfer systems comprising viral elements (e.g., intact viruses,modified viruses and viral components such as nucleic acids or proteins)to facilitate delivery of the sample to a desired cell or tissue. Asused herein, the term “adenovirus gene transfer system” refers to genetransfer systems comprising intact or altered viruses belonging to thefamily Adenoviridae.

As used herein, the term “site-specific recombination target sequences”refers to nucleic acid sequences that provide recognition sequences forrecombination factors and the location where recombination takes place.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of apolypeptide, precursor, or RNA (e.g., rRNA, tRNA). The polypeptide canbe encoded by a full length coding sequence or by any portion of thecoding sequence so long as the desired activity or functional properties(e.g., enzymatic activity, ligand binding, signal transduction,immunogenicity, etc.) of the full-length or fragment are retained. Theterm also encompasses the coding region of a structural gene and thesequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb or more on either end such that thegene corresponds to the length of the full-length mRNA. Sequenceslocated 5′ of the coding region and present on the mRNA are referred toas 5′ non-translated sequences. Sequences located 3′ or downstream ofthe coding region and present on the mRNA are referred to as 3′non-translated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the term “heterologous gene” refers to a gene that isnot in its natural environment. For example, a heterologous geneincludes a gene from one species introduced into another species. Aheterologous gene also includes a gene native to an organism that hasbeen altered in some way (e.g., mutated, added in multiple copies,linked to non-native regulatory sequences, etc). Heterologous genes aredistinguished from endogenous genes in that the heterologous genesequences are typically joined to DNA sequences that are not foundnaturally associated with the gene sequences in the chromosome or areassociated with portions of the chromosome not found in nature (e.g.,genes expressed in loci where the gene is not normally expressed).

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 200 residues long (e.g., between 15 and 100), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 24 residue oligonucleotide is referred to as a “24-mer”.Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence“5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This is of particular importance inamplification reactions, as well as detection methods that depend uponbinding between nucleic acids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is a nucleic acid molecule that at leastpartially inhibits a completely complementary nucleic acid molecule fromhybridizing to a target nucleic acid is “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous nucleic acid molecule to a target underconditions of low stringency. This is not to say that conditions of lowstringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second target that issubstantially non-complementary (e.g., less than about 30% identity); inthe absence of non-specific binding the probe will not hybridize to thesecond non-complementary target.

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described above.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids. A single molecule that contains pairing of complementarynucleic acids within its structure is said to be “self-hybridized.”

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Under “low stringency conditions” anucleic acid sequence of interest will hybridize to its exactcomplement, sequences with single base mismatches, closely relatedsequences (e.g., sequences with 90% or greater homology), and sequenceshaving only partial homology (e.g., sequences with 50-90% homology).Under ‘medium stringency conditions,” a nucleic acid sequence ofinterest will hybridize only to its exact complement, sequences withsingle base mismatches, and closely relation sequences (e.g., 90% orgreater homology). Under “high stringency conditions,” a nucleic acidsequence of interest will hybridize only to its exact complement, and(depending on conditions such a temperature) sequences with single basemismatches. In other words, under conditions of high stringency thetemperature can be raised so as to exclude hybridization to sequenceswith single base mismatches.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1× SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5× SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5× Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0× SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5× SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5× Denhardt's reagent [50× Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5× SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.)(see definition above for “stringency”).

As used herein, the term “amplification oligonucleotide” refers to anoligonucleotide that hybridizes to a target nucleic acid, or itscomplement, and participates in a nucleic acid amplification reaction.An example of an amplification oligonucleotide is a “primer” thathybridizes to a template nucleic acid and contains a 3′ OH end that isextended by a polymerase in an amplification process. Another example ofan amplification oligonucleotide is an oligonucleotide that is notextended by a polymerase (e.g., because it has a 3′ blocked end) butparticipates in or facilitates amplification. Amplificationoligonucleotides may optionally include modified nucleotides or analogs,or additional nucleotides that participate in an amplification reactionbut are not complementary to or contained in the target nucleic acid.Amplification oligonucleotides may contain a sequence that is notcomplementary to the target or template sequence. For example, the 5′region of a primer may include a promoter sequence that isnon-complementary to the target nucleic acid (referred to as a“promoter-primer”). Those skilled in the art will understand that anamplification oligonucleotide that functions as a primer may be modifiedto include a 5′ promoter sequence, and thus function as apromoter-primer. Similarly, a promoter-primer may be modified by removalof, or synthesis without, a promoter sequence and still function as aprimer. A 3′ blocked amplification oligonucleotide may provide apromoter sequence and serve as a template for polymerization (referredto as a “promoter-provider”).

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced, (i.e., in the presence of nucleotides and an inducing agentsuch as DNA polymerase and at a suitable temperature and pH). The primeris preferably single stranded for maximum efficiency in amplification,but may alternatively be double stranded. If double stranded, the primeris first treated to separate its strands before being used to prepareextension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether occurring naturally as in a purifiedrestriction digest or produced synthetically, recombinantly or by PCRamplification, that is capable of hybridizing to at least a portion ofanother oligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the disclosed methods may be labeled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g., ELISA, as well as enzyme-basedhistochemical assays), fluorescent, radioactive, and luminescentsystems. The compositions and methods that use such compositions asdisclosed herein are not limited to any particular detection system orlabel.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecomponent or contaminant with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is such present in a form orsetting that is different from that in which it is found in nature. Incontrast, non-isolated nucleic acids as nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding a given protein includes, by way ofexample, such nucleic acid in cells ordinarily expressing the givenprotein where the nucleic acid is in a chromosomal location differentfrom that of natural cells, or is otherwise flanked by a differentnucleic acid sequence than that found in nature. The isolated nucleicacid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be used to express a protein,the oligonucleotide or polynucleotide will contain at a minimum thesense or coding strand (i.e., the oligonucleotide or polynucleotide maybe single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof components (e.g., contaminants) from a sample. For example,antibodies are purified by removal of contaminating non-immunoglobulinproteins; they are also purified by the removal of immunoglobulin thatdoes not bind to the target molecule. The removal of non-immunoglobulinproteins and/or the removal of immunoglobulins that do not bind to thetarget molecule results in an increase in the percent of target-reactiveimmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed compositions and methods are based on the discovery ofrecurrent gene rearrangements in prostate cancer. These compositions andmethods are useful for diagnostic applications, therapeutic methods, andevaluation of therapeutic treatments that either directly or indirectlydetect or target MIPOL1-ETV1 gene rearrangements.

I. Gene Rearrangements

Recurrent gene rearrangements disclosed herein are indicative ofprostate cancer. These genetic rearrangements result from chromosomalrearrangements that insert MIPOL1 genetic material into the ETV1 geneticlocus. These recurrent gene rearrangements are useful diagnostic markersand clinical targets for prostate cancer.

In some embodiments, all or a portion of the ETV1 is inserted into theMIPOL1 locus (e.g., into an intron). The gene rearrangement isdetectable, for example, as a chromosomal rearrangement of genomic DNAhaving at least a portion of an ETV1 gene inserted into a MIPOL1 locus.

II. Antibodies

The gene rearrangements disclosed herein result in proteins, whichinclude fragments, derivatives and analogs thereof, which may be used asimmunogens to produce antibodies useful for diagnostic and therapeuticapplications. Such antibodies may be polyclonal or monoclonal, chimeric,humanized, single chain or Fab fragments, which may be labeled orunlabeled, all of which may be produced by using well known proceduresand standard laboratory practices. See, e.g., Burns, ed., ImmunochemicalProtocols, 3^(rd) ed., Humana Press (2005); Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory (1988); Kozbor etal., Immunology Today 4: 72 (1983); Köhler and Milstein, Nature 256: 495(1975).

III. Diagnostic Applications

The disclosed MIPOL1-ETV1 genetic rearrangements provide DNA, RNA andprotein based diagnostic methods that detect, either directly orindirectly, the gene rearrangements or a product made specifically as aresult of the genetic rearrangement. The disclosed MIPOL1-ETV1 geneticrearrangements also provide compositions useful for diagnostic purposes,such as oligonucleotide probes that specifically detect all or part ofthe genetic rearrangement. Such compositions may be in the form of akit.

The disclosed diagnostic methods may be qualitative or quantitative.Quantitative methods may, e.g., discriminate between indolent andaggressive cancers via a cutoff or threshold level where expressionabove that level provides information on the aggressiveness of thecancer which provides useful diagnostic and/or prognostic information toa treating physician or patient. Qualitative or quantitative diagnosticmethods may include amplification of a target, signal or intermediary,such as by using a universal primer that amplifies a sequence thatserves as an indicator for the presence or level of the specific targetassociated with the MIPOL1-ETV1 genetic rearrangement.

An initial assay may confirm the presence of a gene rearrangement butnot identify the specific rearrangement. A secondary assay is thenperformed to determine the identity of the particular rearrangement, ifdesired. The second assay may use a different detection technology thanthe initial assay.

The disclosed MIPOL1-ETV1 genetic rearrangement may be detected alongwith other markers in a multiplex or panel format. Markers are selectedfor their predictive value alone or in combination with the generearrangements. Exemplary prostate cancer markers include, but are notlimited to: AMACR/P504S (U.S. Pat. No. 6,262,245); PCA3 (U.S. Pat. No.7,008,765); PCGEM1 (U.S. Pat. No. 6,828,429); prostein/P501S, P503S,P504S, P509S, P510S, prostase/P703P, P710P (U.S. Publication No.20030185830); and, those disclosed in U.S. Pat. Nos. 5,854,206 and6,034,218, and U.S. Publication No. 20030175736, each of which is hereinincorporated by reference in its entirety. Markers for other cancers,diseases, infections, and metabolic conditions are also contemplated forinclusion in a multiplex of panel format.

The diagnostic methods as disclosed herein may be modified withreference to data correlating particular gene rearrangements with thestage, aggressiveness or progression of the disease or the presence orrisk of metastasis. The information provided by these diagnostic methodsprovide useful information to a physician who, based on thatinformation, may choose an appropriate therapeutic treatment for aparticular patient.

A. Sample

Any biological sample suspected of containing the MIPOL1-ETV1 generearrangements may be tested according to the disclosed methods. Such asample may be tissue (e.g., prostate biopsy sample or tissue obtained byprostatectomy), blood, urine, semen, prostatic secretions or a fractionthereof (e.g., plasma, serum, urine supernatant, urine cell pellet orprostate cells), which may be obtained from a patient or other source ofbiological material, e.g., autopsy sample or forensic material. Inpreferred embodiments, a urine sample is collected immediately followingan attentive digital rectal examination (DRE), which causes prostatecells from the prostate gland to shed into the urinary tract.

The sample may be processed to isolate or enrich the sample for the generearrangements or cells that contain the gene rearrangements. A varietyof well techniques that use standard laboratory practices may be usedfor this purpose, such as, e.g., centrifugation, immunocapture, celllysis, and nucleic acid target capture (See, e.g., EP Pat. No. 1 409727, herein incorporated by reference in its entirety).

B. DNA and RNA Detection

The disclosed MIPOL1-ETV1 genetic rearrangements may be detected aschromosomal rearrangements of genomic DNA or as chimeric mRNA producedfrom a chromosomal rearrangement by using a variety of well knownnucleic acid techniques that rely on standard laboratory methods, suchas, e.g., nucleic acid sequencing, nucleic acid hybridization, and,nucleic acid amplification.

1. Sequencing

Illustrative non-limiting examples of nucleic acid sequencing techniquesinclude, but are not limited to, chain terminator (Sanger) sequencingand dye terminator sequencing. Those of ordinary skill in the art willrecognize that because RNA is less stable in the cell and more prone tonuclease attack experimentally RNA is usually reverse transcribed to DNAbefore sequencing.

Chain terminator sequencing uses sequence-specific termination of a DNAsynthesis reaction using modified nucleotide substrates. Extension isinitiated at a specific site on the template DNA by using a shortradioactive, or other labeled, oligonucleotide primer complementary tothe template at that region. The oligonucleotide primer is extendedusing a DNA polymerase, standard four deoxynucleotide bases, and a lowconcentration of one chain terminating nucleotide, most commonly adi-deoxynucleotide. This reaction is repeated in four separate tubeswith each of the bases taking turns as the di-deoxynucleotide. Limitedincorporation of the chain terminating nucleotide by the DNA polymeraseresults in a series of related DNA fragments that are terminated only atpositions where that particular di-deoxynucleotide is used. For eachreaction tube, the fragments are size-separated by electrophoresis in aslab polyacrylamide gel or a capillary tube filled with a viscouspolymer. The sequence is determined by reading which lane produces avisualized mark from the labeled primer as you scan from the top of thegel to the bottom.

Dye terminator sequencing alternatively labels the terminators. Completesequencing can be performed in a single reaction by labeling each of thedi-deoxynucleotide chain-terminators with a separate fluorescent dye,which fluoresces at a different wavelength.

2. Hybridization

Illustrative non-limiting examples of nucleic acid hybridizationtechniques include, but are not limited to, in situ hybridization (ISH),microarray, and Southern or Northern blot.

In situ hybridization (ISH) is a type of hybridization that uses alabeled complementary DNA or RNA strand as a probe to localize aspecific DNA or RNA sequence in a portion or section of tissue (insitu), or, if the tissue is small enough, the entire tissue (whole mountISH). DNA ISH can be used to determine the structure of chromosomes. RNAISH is used to measure and localize mRNAs and other transcripts withintissue sections or whole mounts. Sample cells and tissues are usuallytreated to fix the target transcripts in place and to increase access ofthe probe. The probe hybridizes to the target sequence at elevatedtemperature, and then the excess probe is washed away. The probe thatwas labeled with either radio-, fluorescent- or antigen-labeled bases islocalized and quantitated in the tissue using either autoradiography,fluorescence microscopy or immunohistochemistry, respectively. ISH canalso use two or more probes, labeled with radioactivity or the othernon-radioactive labels, to simultaneously detect two or moretranscripts.

2.1 FISH

In some embodiments, gene rearrangements are detected using fluorescencein situ hybridization (FISH). The preferred FISH assays use bacterialartificial chromosomes (BACs), which have been used extensively in thehuman genome sequencing project (see Nature 409: 953-958 (2001)) andclones containing specific BACs are widely available or can be made byusing standard laboratory practices. Each BAC clone from the humangenome has been given a reference name that unambiguously identifies it.These names can be used to find a corresponding GenBank sequence and toorder copies of the clone from a distributor.

Specific BAC clones that can be used in FISH protocols to detectMIPOL1-ETV1 genetic rearrangements are probes specific for RP11-124L22and RP11-703A4. These probes, or other suitable probes, usually arelabeled with appropriate fluorescent or other markers and then used inhybridizations. The Examples section provided herein sets forth oneparticular protocol that is effective for measuring rearrangements butone of skill in the art will recognize that many variations of thisassay can be used equally well. Specific protocols are well known in theart and can be readily adapted for detecting MIPOL1-ETV1 rearrangements.Guidance regarding such methodology is provided in many referencesincluding: In situ Hybridization: Medical Applications (eds. G. R.Coulton and J. de Belleroche), Kluwer Academic Publishers, Boston(1992); In situ Hybridization: In Neurobiology; Advances in Methodology(eds. J. H. Eberwine, K. L. Valentino, and J. D. Barchas), OxfordUniversity Press Inc., England (1994); In situ Hybridization: APractical Approach (ed. D. G. Wilkinson), Oxford University Press Inc.,England (1992)); Kuo, et al., Am. J. Hum. Genet. 49:112-119 (1991);Klinger, et al., Am. J. Hum. Genet. 51:55-65 (1992); and Ward, et al.,Am. J. Hum. Genet. 52:854-865 (1993)). Patents providing guidance onsuch methodology include U.S. Pat. Nos. 5,225,326; 5,545,524; 6,121,489and 6,573,043, and commercially available kits also provide protocolsfor performing FISH (e.g., from Oncor, Inc., Gaithersburg, Md.). All ofthese references are hereby incorporated by reference in their entiretyand may be used along with similar references in the art and with theinformation provided in the Examples section herein to establishprocedural steps convenient for a particular laboratory.

2.2 Microarrays

Different kinds of biological assays are called microarrays including,but not limited to: DNA microarrays (e.g., cDNA microarrays andoligonucleotide microarrays); protein microarrays; tissue microarrays;transfection or cell microarrays; chemical compound microarrays; and,antibody microarrays. A DNA microarray, commonly known as gene chip, DNAchip, or biochip, is a collection of microscopic DNA spots attached to asolid surface (e.g., glass, plastic or silicon chip) forming an arrayfor the purpose of expression profiling or monitoring expression levelsfor thousands of genes simultaneously. The affixed DNA segments areknown as probes, thousands of which can be used in a single DNAmicroarray. Microarrays can be used to identify disease genes bycomparing gene expression in disease and normal cells. Microarrays canbe fabricated using a variety of technologies, including but notlimiting: printing with fine-pointed pins onto glass slides;photolithography using pre-made masks; photolithography using dynamicmicromirror devices; ink-jet printing; or, electrochemistry onmicroelectrode arrays.

Southern and Northern blotting is used to detect specific DNA or RNAsequences, respectively. DNA or RNA extracted from a sample isfragmented, electrophoretically separated on a matrix gel, andtransferred to a membrane filter. The filter bound DNA or RNA is subjectto hybridization with a labeled probe complementary to the sequence ofinterest. Hybridized probe bound to the filter is detected. A variant ofthe procedure is the reverse Northern blot, in which the substratenucleic acid that is affixed to the membrane is a collection of isolatedDNA fragments and the probe is RNA extracted from a tissue and labeled.

3. Amplification

Chromosomal rearrangements of genomic DNA and chimeric mRNA may beamplified prior to or simultaneous with detection. Illustrativenon-limiting examples of nucleic acid amplification techniques include,but are not limited to, polymerase chain reaction (PCR), reversetranscription polymerase chain reaction (RT-PCR), transcription-mediatedamplification (TMA), ligase chain reaction (LCR), strand displacementamplification (SDA), and nucleic acid sequence based amplification(NASBA). Those of ordinary skill in the art will recognize that certainamplification techniques (e.g., PCR) require that RNA be reversedtranscribed to DNA prior to amplification (e.g., RT-PCR), whereas otheramplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159 and 4,965,188, each of which is herein incorporated byreference in its entirety), commonly referred to as PCR, uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to exponentially increase copy numbers of a targetnucleic acid sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.For other various permutations of PCR see, e.g., U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155:335 (1987); and, Murakawa et al., DNA 7: 287 (1988), each of which isherein incorporated by reference in its entirety.

Transcription mediated amplification (U.S. Pat. Nos. 5,480,784 and5,399,491, each of which is herein incorporated by reference in itsentirety), commonly referred to as TMA, synthesizes multiple copies of atarget nucleic acid sequence autocatalytically under conditions ofsubstantially constant temperature, ionic strength, and pH in whichmultiple RNA copies of the target sequence autocatalytically generateadditional copies. See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518,each of which is herein incorporated by reference in its entirety. In avariation described in U.S. Publ. No. 20060046265 (herein incorporatedby reference in its entirety), TMA optionally incorporates the use ofblocking moieties, terminating moieties, and other modifying moieties toimprove TMA process sensitivity and accuracy.

The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), hereinincorporated by reference in its entirety), commonly referred to as LCR,uses two sets of complementary DNA oligonucleotides that hybridize toadjacent regions of the target nucleic acid. The DNA oligonucleotidesare covalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad.Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455,166,each of which is herein incorporated by reference in its entirety),commonly referred to as SDA, uses cycles of annealing pairs of primersequences to opposite strands of a target sequence, primer extension inthe presence of a dNTPαS to produce a duplex hemiphosphorothioatedprimer extension product, endonuclease-mediated nicking of ahemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (EP Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequencebased amplification (U.S. Pat. No. 5,130,238, herein incorporated byreference in its entirety), commonly referred to as NASBA; one that usesan RNA replicase to amplify the probe molecule itself (Lizardi et al.,BioTechnol. 6: 1197 (1988), herein incorporated by reference in itsentirety), commonly referred to as Qβ replicase; a transcription basedamplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173(1989)); and, self-sustained sequence replication (Guatelli et al.,Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is hereinincorporated by reference in its entirety). For further discussion ofknown amplification methods see Persing, David H., “In Vitro NucleicAcid Amplification Techniques” in Diagnostic Medical Microbiology:Principles and Applications (Persing et al., Eds.), pp. 51-87 (AmericanSociety for Microbiology, Washington, D.C. (1993)).

4. Detection Methods

Non-amplified or amplified gene rearrangement nucleic acids can bedetected by any conventional means. For example, the gene rearrangementscan be detected by hybridization with a detectably labeled probe andmeasurement of the resulting hybrids. Illustrative non-limiting examplesof detection methods are described below.

One illustrative detection method, the Hybridization Protection Assay(HPA) involves hybridizing a chemiluminescent oligonucleotide probe(e.g., an acridinium ester-labeled (AE) probe) to the target sequence,selectively hydrolyzing the chemiluminescent label present onunhybridized probe, and measuring the chemiluminescence produced fromthe remaining probe in a luminometer. See, e.g., U.S. Pat. No. 5,283,174and Norman C. Nelson et al., Nonisotopic Probing, Blotting, andSequencing, ch. 17 (Larry J. Kricka ed., 2d ed. 1995, each of which isherein incorporated by reference in its entirety).

Another illustrative detection method provides for quantitativeevaluation of the amplification process in real-time. Evaluation of anamplification process in “real-time” involves determining the amount ofamplicon in the reaction mixture either continuously or periodicallyduring the amplification reaction, and using the determined values tocalculate the amount of target sequence initially present in the sample.A variety of methods for determining the amount of initial targetsequence present in a sample based on real-time amplification are wellknown in the art. These include methods disclosed in U.S. Pat. Nos.6,303,305 and 6,541,205, each of which is herein incorporated byreference in its entirety. Another method for determining the quantityof target sequence initially present in a sample, but which is not basedon a real-time amplification, is disclosed in U.S. Pat. No. 5,710,029,herein incorporated by reference in its entirety.

Amplification products may be detected in real-time through the use ofvarious self-hybridizing probes, most of which have a stem-loopstructure. Such self-hybridizing probes are labeled so that they emitdifferently detectable signals, depending on whether the probes are in aself-hybridized state or an altered state through hybridization to atarget sequence. By way of non-limiting example, “molecular torches” area type of self-hybridizing probe that includes distinct regions ofself-complementarity (referred to as “the target binding domain” and“the target closing domain”) which are connected by a joining region(e.g., non-nucleotide linker) and which hybridize to each other underpredetermined hybridization assay conditions. In a preferred embodiment,molecular torches contain single-stranded base regions in the targetbinding domain that are from 1 to about 20 bases in length and areaccessible for hybridization to a target sequence present in anamplification reaction under strand displacement conditions. Understrand displacement conditions, hybridization of the two complementaryregions, which may be fully or partially complementary, of the moleculartorch is favored, except in the presence of the target sequence, whichwill bind to the single-stranded region present in the target bindingdomain and displace all or a portion of the target closing domain. Thetarget binding domain and the target closing domain of a molecular torchinclude a detectable label or a pair of interacting labels (e.g.,luminescent/quencher) positioned so that a different signal is producedwhen the molecular torch is self-hybridized than when the moleculartorch is hybridized to the target sequence, thereby permitting detectionof probe:target duplexes in a test sample in the presence ofunhybridized molecular torches. Molecular torches and many types ofinteracting label pairs are known (e.g., U.S. Pat. No. 6,534,274, hereinincorporated by reference in its entirety).

Another example of a detection probe having self-complementarity is a“molecular beacon” (see U.S. Pat. Nos. 5,925,517 and 6,150,097, hereinincorporated by reference in entirety). Molecular beacons includenucleic acid molecules having a target complementary sequence, anaffinity pair (or nucleic acid arms) holding the probe in a closedconformation in the absence of a target sequence present in anamplification reaction, and a label pair that interacts when the probeis in a closed conformation. Hybridization of the target sequence andthe target complementary sequence separates the members of the affinitypair, thereby shifting the probe to an open conformation. The shift tothe open conformation is detectable due to reduced interaction of thelabel pair, which may be, for example, a fluorophore and a quencher(e.g., DABCYL and EDANS).

Other self-hybridizing probes are well known to those of ordinary skillin the art. By way of non-limiting example, probe binding pairs havinginteracting labels (e.g., see U.S. Pat. No. 5,928,862, hereinincorporated by reference in its entirety) may be adapted for use in thecompositions and methods disclosed herein. Probe systems used to detectsingle nucleotide polymorphisms (SNPs) might also be used. Additionaldetection systems include “molecular switches,” (e.g., see U.S. Publ.No. 20050042638, herein incorporated by reference in its entirety).Other probes, such as those comprising intercalating dyes and/orfluorochromes, are also useful for detection of amplification productsin the methods disclosed herein (e.g., see U.S. Pat. No. 5,814,447,herein incorporated by reference in its entirety).

C. Data Analysis

In some embodiments, a computer-based analysis program is used totranslate the raw data generated by the detection assay (e.g., thepresence, absence, or amount of a given marker or markers) into data ofpredictive value for a clinician. The clinician can access thepredictive data using any suitable means. Thus, in some preferredembodiments, the present invention provides the further benefit that theclinician, who is not likely to be trained in genetics or molecularbiology, need not understand the raw data. The data is presenteddirectly to the clinician in its most useful form. The clinician is thenable to immediately utilize the information in order to optimize thecare of the subject.

Any method may be used that is capable of receiving, processing, andtransmitting the information to and from laboratories conducting theassays, information provides, medical personal, and subjects. Forexample, in some embodiments of the present invention, a sample (e.g., abiopsy or a serum or urine sample) is obtained from a subject andsubmitted to a profiling service (e.g., clinical lab at a medicalfacility, genomic profiling business, etc.), located in any part of theworld (e.g., in a country different than the country where the subjectresides or where the information is ultimately used) to generate rawdata. Where the sample comprises a tissue or other biological sample,the subject may visit a medical center to have the sample obtained andsent to the profiling center, or subjects may collect the samplethemselves (e.g., a urine sample) and directly send it to a profilingcenter. Where the sample comprises previously determined biologicalinformation, the information may be directly sent to the profilingservice by the subject (e.g., an information card containing theinformation may be scanned by a computer and the data transmitted to acomputer of the profiling center using an electronic communicationsystems). Once received by the profiling service, the sample isprocessed and a profile is produced (i.e., expression data), specificfor the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable forinterpretation by a treating clinician. For example, rather thanproviding raw expression data, the prepared format may represent adiagnosis or risk assessment (e.g., likelihood of cancer being present)for the subject, along with recommendations for particular treatmentoptions. The data may be displayed to the clinician by any suitablemethod. For example, in some embodiments, the profiling servicegenerates a report that can be printed for the clinician (e.g., at thepoint of care) or displayed to the clinician on a computer monitor.

In some embodiments, the information is first analyzed at the point ofcare or at a regional facility. The raw data is then sent to a centralprocessing facility for further analysis and/or to convert the raw datato information useful for a clinician or patient. The central processingfacility provides the advantage of privacy (all data is stored in acentral facility with uniform security protocols), speed, and uniformityof data analysis. The central processing facility can then control thefate of the data following treatment of the subject. For example, usingan electronic communication system, the central facility can providedata to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the datausing the electronic communication system. The subject may chose furtherintervention or counseling based on the results. In some embodiments,the data is used for research use. For example, the data may be used tofurther optimize the inclusion or elimination of markers as usefulindicators of a particular condition or stage of disease.

D. In Vivo Imaging

The gene rearrangements disclosed herein may also be detected using invivo imaging techniques, including but not limited to: radionuclideimaging; positron emission tomography (PET); computerized axialtomography, X-ray or magnetic resonance imaging method, fluorescencedetection, and chemiluminescent detection. In some embodiments, in vivoimaging techniques are used to visualize the presence of or expressionof cancer markers in an animal (e.g., a human or non-human mammal). Forexample, in some embodiments, cancer marker mRNA or protein is labeledusing a labeled antibody specific for the cancer marker. A specificallybound and labeled antibody can be detected in an individual using an invivo imaging method, including, but not limited to, radionuclideimaging, positron emission tomography, computerized axial tomography,X-ray or magnetic resonance imaging method, fluorescence detection, andchemiluminescent detection. Methods for generating antibodies to thedisclosed cancer markers are described above.

The in vivo imaging methods that use the compositions disclosed hereinthat detect MIPOL1-ETV1 rearrangements or products derived from them areuseful in the diagnosis of cancers, particularly prostate cancer, thatexpress the cancer markers disclosed herein. In vivo imaging visualizesthe presence of a marker indicative of the cancer, allowing diagnosisand/or prognosis without the use of an unpleasant biopsy. For example,the presence of a marker indicative of cancers likely to metastasize canbe detected. The in vivo imaging methods can further be used to detectmetastatic cancers in other parts of the body.

In some embodiments, reagents (e.g., antibodies) specific for the cancermarkers associated with MIPOL1-ETV1 genetic rearrangements arefluorescently labeled. The labeled antibodies are introduced into asubject (e.g., orally or parenterally). Fluorescently labeled antibodiesare detected using any suitable method or system (e.g., see U.S. Pat.No. 6,198,107, herein incorporated by reference).

In other embodiments, antibodies are radioactively labeled. The use ofantibodies for in vivo diagnosis is well known in the art, e.g., byusing an antibody-based labeling system to image tumors (see Sumerdon etal., Nucl. Med. Biol 17:247-254 [1990], Griffin et al., J. Clin. Onc.9:631-640 [1991], and Lauffer, Magnetic Resonance in Medicine 22:339-342[1991]). The label used with an antibody-based system will depend on theimaging modality chosen, for example, radioactive labels such asIndium-111, Technetium-99m, or Iodine-131 for use with planar scans orsingle photon emission computed tomography (SPECT), positron emittinglabels such as Fluorine-19 for use with positron emission tomography(PET), and paramagnetic ions such as Gadolinium (III) or Manganese (II)for use with MRI.

Radioactive metals with half-lives ranging from 1 hour to 3.5 days areavailable for conjugation to antibodies, such as scandium-47 (3.5 days)gallium-67 (2.8 days), gallium-68 (68 minutes), technetium-99m (6hours), and indium-111 (3.2 days), of which gallium-67, technetium-99m,and indium-111 are preferable for gamma camera imaging, gallium-68 ispreferable for positron emission tomography.

A useful method of labeling antibodies with such radiometals is by meansof a bifunctional chelating agent, such as diethylenetriaminepentaaceticacid (DTPA), as described, for example, by Khaw et al. (Science 209:295[1980]) for In-111 and Tc-99m, and by Scheinberg et al. (Science215:1511 [1982]). Other chelating agents may also be used, but the1-(p-carboxymethoxybenzyl)EDTA and the carboxycarbonic anhydride of DTPAare advantageous because their use permits conjugation without affectingthe antibody's immunoreactivity substantially.

Another method for coupling DPTA to proteins is by use of the cyclicanhydride of DTPA, as described by Hnatowich et al. (Int. J. Appl.Radiat. Isot. 33:327 [1982]) for labeling of albumin with In-111, butwhich can be adapted for labeling of antibodies. A suitable method oflabeling antibodies with Tc-99m is known (e.g., see Crockford et al.,U.S. Pat. No. 4,323,546, herein incorporated by reference).

A preferred method of labeling immunoglobulins with Tc-99m is thatdescribed by Wong et al. (Int. J. Appl. Radiat. Isot., 29:251 [1978])for plasma protein, and recently applied successfully by Wong et al. (J.Nucl. Med., 23:229 [1981]) for labeling antibodies.

In the case of the radiometals conjugated to the specific antibody, itis likewise desirable to introduce as high a proportion of theradiolabel as possible into the antibody molecule without destroying itsimmunospecificity. A further improvement may be achieved by effectingradiolabeling in the presence of the specific cancer marker associatedwith a MIPOL1-ETV1 genetic rearrangement, to insure that the antigenbinding site on the antibody is protected. The antigen is separatedafter labeling.

In still further embodiments, in vivo biophotonic imaging (Xenogen,Almeda, Calif.) is used for in vivo imaging. This real-time in vivoimaging utilizes luciferase, an enzyme that catalyzes a light-emittingreaction. The luciferase gene is incorporated into cells,microorganisms, and animals (e.g., to produce a fusion protein with acancer marker associated with a MIPOL1-ETV1 genetic rearrangement), sothat when the cancer marker is active a light emission occurs which iscaptured as an image and analyzed by using a CCD camera and appropriatesoftware.

E. Compositions & Kits

Compositions for use in the disclosed diagnostic methods include, butare not limited to, probes, amplification oligonucleotides, andantibodies. The compositions detect a product only when a MIPOL1-ETV1rearrangement is present, preferably ETV1 genetic material inserted intoMIPOL1 genetic material. These compositions include, but are not limitedto: a single labeled probe comprising a sequence that hybridizes to thejunction at which ETV1 is inserted into the MIPOL1 locus (i.e., spansthe gene rearrangement junction); a pair of amplificationoligonucleotides wherein the first amplification oligonucleotidecomprises a sequence that hybridizes to MIPOL1 and a secondamplification oligonucleotide comprising a sequence that hybridizes toETV1.

Other useful compositions, however, include: a pair of labeled probeswherein the first labeled probe comprises a sequence that hybridizes toMIPOL1 and the second labeled probe comprises a sequence that hybridizesto ETV1.

Any of these compositions, alone or in combination with othercompositions disclosed herein or well known in the art, may be providedin the form of a kit. For example, the single labeled probe and pair ofamplification oligonucleotides may be provided in a kit for theamplification and detection of MIPOL1-ETV1 genetic rearrangements. Kitsmay further comprise appropriate controls and/or detection reagents. Anyone or more reagents that find use in any of the methods describedherein may be provided in the kit.

The probe and antibody compositions may also be provided in the form ofan array.

IV. Drug Screening Applications

In some embodiments, the disclosed compositions and methods are used indrug screening assays (e.g., to screen for anticancer drugs). Thesescreening methods use cancer markers that include those associated withMIPOL1-ETV1 gene rearrangements, but are not limited only to thosegenetic rearrangements. For example, an embodiment may screen forcompounds that alter (e.g., decrease) the expression of cancer markergenes, including those associated with MIPOL1-ETV1 gene rearrangements.Compounds or agents to be screened for may interfere with transcription(e.g., by interacting with a promoter region), may interfere with mRNAproduced from the rearrangement (e.g., by RNA interference, antisensetechnologies, etc.), or may interfere with pathways that are upstream ordownstream of the biological activity of the gene rearrangement. In someembodiments, candidate compounds are antisense or interfering RNA agents(e.g., oligonucleotides) directed against cancer markers. In otherembodiments, candidate compounds are antibodies or small molecules thatspecifically bind to a cancer marker regulator or expression productassociated with MIPOL1-ETV1 gene rearrangements and inhibit itsbiological function.

In some embodiments, candidate compounds are evaluated for their abilityto alter cancer marker expression by contacting a compound with a cellexpressing a cancer marker and then assaying for the effect of thecandidate compounds on expression. In some embodiments, the effect ofcandidate compounds on expression of a cancer marker gene is assayed forby detecting the level of cancer marker mRNA expressed by the cell. mRNAexpression can be detected by any suitable method. In other embodiments,the effect of candidate compounds on expression of cancer marker genesis assayed by measuring the level of polypeptide encoded by the cancermarkers. The level of polypeptide expressed can be measured using anysuitable method, including but not limited to, those disclosed herein.

The test compounds can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including biologicallibraries; peptoid libraries (libraries of molecules having thefunctionalities of peptides, but with a novel, non-peptide backbone,which are resistant to enzymatic degradation but which neverthelessremain bioactive; see, e.g., Zuckennann et al., J. Med. Chem. 37:2678-85 [1994]); spatially addressable parallel solid phase or solutionphase libraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library andpeptoid library approaches are preferred for use with peptide libraries,while the other four approaches are applicable to peptide, non-peptideoligomer or small molecule libraries of compounds (Lam (1997) AnticancerDrug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al., Proc. Natl. Acad. Sci.U.S.A. 90:6909 [1993]; Erb et al., Proc. Nad. Acad. Sci. USA 91:11422[1994]; Zuckermann et al., J. Med. Chem. 37:2678 [1994]; Cho et al.,Science 261:1303 [1993]; Carrell et al., Angew. Chem. Int. Ed. Engl.33.2059 [1994]; Carell et al., Angew. Chem. Int. Ed. Engl. 33:2061[1994]; and Gallop et al., J. Med. Chem. 37:1233 [1994].

Libraries of compounds may be presented in solution (e.g., Houghten,Biotechniques 13:412-421 [1992]), or on beads (Lam, Nature 354:82-84[1991]), chips (Fodor, Nature 364:555-556 [1993]), bacteria or spores(U.S. Pat. No. 5,223,409; herein incorporated by reference), plasmids(Cull et al., Proc. Nad. Acad. Sci. USA 89:18651869 [1992]) or on phage(Scott and Smith, Science 249:386-390 [1990]; Devlin Science 249:404-406[1990]; Cwirla et al., Proc. NatI. Acad. Sci. 87:6378-6382 [1990];Felici, J. Mol. Biol. 222:301 [1991]).

VI. Therapeutic Applications

Some embodiments provide therapies for cancer (e.g., prostate cancer).Preferred therapy embodiments target directly or indirectly cancermarkers, including those associated with MIPOL1-ETV1 geneticrearrangements.

A. RNA Interference and Antisense Therapies

Some embodiments target the expression of cancer markers associated withMIPOL1-ETV1 genetic rearrangements. Some embodiments employ compositionscomprising oligomeric antisense or RNAi compounds, particularlyoligonucleotides (e.g., those identified in the drug screening methodsdescribed above), for use in modulating the function of nucleic acidmolecules encoding cancer markers associated with MIPOL1-ETV1 geneticrearrangements, ultimately modulating the amount of cancer markerexpressed.

1. RNA Interference (RNAi)

In some embodiments, RNAi is used to inhibit expression of MIPOL1-ETV1gene rearrangements. RNAi represents an evolutionary conserved cellulardefense for controlling the expression of foreign genes in mosteukaryotes, including humans. RNAi is typically triggered bydouble-stranded RNA (dsRNA) and causes sequence-specific mRNAdegradation of single-stranded target RNAs homologous in response todsRNA. The mediators of mRNA degradation are small interfering RNAduplexes (siRNAs), which are normally produced from long dsRNA byenzymatic cleavage in the cell. siRNAs are generally approximatelytwenty-one nucleotides in length (e.g. 21-23 nucleotides in length), andhave a base-paired structure characterized by two nucleotide3′-overhangs. Following the introduction of a small RNA, or RNAi, intothe cell, it is believed the sequence is delivered to an enzyme complexcalled RISC (RNA-induced silencing complex). RISC recognizes the targetand cleaves it with an endonuclease. It is noted that if larger RNAsequences are delivered to a cell, RNase III enzyme (Dicer) convertslonger dsRNA into 21-23 nt ds siRNA fragments. In some embodiments, RNAioligonucleotides are designed to target the junction region of generearrangements.

Chemically synthesized siRNAs have become powerful reagents forgenome-wide analysis of mammalian gene function in cultured somaticcells. Beyond their value for validation of gene function, siRNAs alsohold great potential as gene-specific therapeutic agents (Tuschl andBorkhardt, Molecular Intervent. 2002; 2(3):158-67, herein incorporatedby reference).

The transfection of siRNAs into animal cells results in the potent,long-lasting post-transcriptional silencing of specific genes (Caplen etal, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al., Nature.2001; 411:494-8; Elbashir et al., Genes Dev. 2001; 15: 188-200; andElbashir et al., EMBO J. 2001; 20: 6877-88, all of which are hereinincorporated by reference). Methods and compositions for performing RNAiwith siRNAs are known (e.g., see U.S. Pat. No. 6,506,559, hereinincorporated by reference).

siRNAs are extraordinarily effective at lowering the amounts of targetedRNA, and by extension proteins, frequently to undetectable levels. Thesilencing effect can last several months, and is extraordinarilyspecific, because one nucleotide mismatch between the target RNA and thecentral region of the siRNA is frequently sufficient to preventsilencing (Brummelkamp et al, Science 2002; 296:550-3; and Holen et al,Nucleic Acids Res. 2002; 30:1757-66, both of which are hereinincorporated by reference).

An important factor in the design of siRNAs is the presence ofaccessible sites for siRNA binding. Bahoia et al., (J. Biol. Chem.,2003; 278: 15991-15997; herein incorporated by reference) describe theuse of a type of DNA array called a scanning array to find accessiblesites in mRNAs for designing effective siRNAs. These arrays compriseoligonucleotides ranging in size from monomers to a certain maximum,usually Comers, synthesized using a physical barrier (mask) by stepwiseaddition of each base in the sequence. Thus the arrays represent a fulloligonucleotide complement of a region of the target gene. Hybridizationof the target mRNA to these arrays provides an exhaustive accessibilityprofile of this region of the target mRNA. Such data are useful in thedesign of antisense oligonucleotides (ranging from 7mers to 25mers),where it is important to achieve a compromise between oligonucleotidelength and binding affinity, to retain efficacy and target specificity(Sohail et al, Nucleic Acids Res., 2001; 29(10): 2041-2045). Additionalmethods and concerns for selecting siRNAs are described for example, inWO 05054270, WO05038054A1, WO03070966A2, J Mol Biol. 2005 May 13;348(4):883-93, J Mol Biol. 2005 May 13; 348(4):871-81, and Nucleic AcidsRes. 2003 Aug. 1; 31(15):4417-24, each of which is herein incorporatedby reference in its entirety. In addition, software (e.g., the MWGonline siMAX siRNA design tool) is commercially or publicly availablefor use in the selection of siRNAs.

2. Antisense

In other embodiments, expression of MIPOL1-ETV1 gene rearrangements ismodulated using antisense compounds that specifically hybridize with oneor more nucleic acids encoding cancer markers associated withMIPOL1-ETV1 genetic rearrangements. The specific hybridization of anoligomeric compound with its target nucleic acid interferes with thenormal function of the nucleic acid. This modulation of function of atarget nucleic acid by compounds that specifically hybridize to it isgenerally referred to as “antisense.” The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity that may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression of cancermarkers associated with MIPOL1-ETV1 genetic rearrangements. Herein,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. For example, expression may beinhibited to potentially prevent tumor proliferation.

Antisense methods preferably target specific nucleic acids. “Targeting”an antisense compound to a particular nucleic acid usually refers to amultistep process that begins with identification of a nucleic acidsequence whose function is to be modulated. This may be, e.g., acellular gene (or mRNA transcribed from the gene) whose expression isassociated with a particular disorder or disease state, or a nucleicacid molecule from an infectious agent. Herein, the target is a nucleicacid molecule encoding a cancer marker associated with MIPOL1-ETV1genetic rearrangements. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Herein, apreferred intragenic site is the region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of thegene. Since the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). Eukaryotic and prokaryotic genes mayhave two or more alternative start codons, any one of which may bepreferentially used for translation initiation in a particular cell typeor tissue, or under a particular set of conditions. Herein, “startcodon” and “translation initiation codon” refer to the codon or codonsthat are used in vivo to initiate translation of an mRNA moleculetranscribed from a gene encoding a tumor antigen associated withMIPOL1-ETV1 genetic rearrangements, regardless of the sequence(s) ofsuch codons.

Translation termination codon (or “stop codon”) of a gene may have oneof three sequences (i.e., 5′-UAA, 5′-UAG and 5′-UGA; the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms“start codon region” and “translation initiation codon region” refer toa portion of such an mRNA or gene that encompasses from about 25 toabout 50 contiguous nucleotides in either direction (i.e., 5′ or 3′)from a translation initiation codon. Similarly, the terms “stop codonregion” and “translation termination codon region” refer to a portion ofsuch an mRNA or gene that encompasses from about 25 to about 50contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon.

The open reading frame (ORF) or “coding region,” which refers to theregion between the translation initiation codon and the translationtermination codon, is also a region that may be targeted effectively.Other target regions include the 5′ untranslated region (5′ UTR),referring to the portion of an mRNA in the 5′ direction from thetranslation initiation codon, and thus including nucleotides between the5′ cap site and the translation initiation codon of an mRNA orcorresponding nucleotides on the gene, and the 3′ untranslated region(3′ UTR), referring to the portion of an mRNA in the 3′ direction fromthe translation termination codon, and thus including nucleotidesbetween the translation termination codon and 3′ end of an mRNA orcorresponding nucleotides on the gene. The 5′ cap of an mRNA comprisesan N7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” that are excised from atranscript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites (i.e., intron-exonjunctions) may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

In some embodiments, target sites for antisense inhibition areidentified using commercially available software programs (e.g.,Biognostik, Gottingen, Germany; SysArris Software, Bangalore, India;Antisense Research Group, University of Liverpool, Liverpool, England;GeneTrove, Carlsbad, Calif.). In other embodiments, target sites forantisense inhibition are identified using the accessible site methoddescribed in PCT Publ. No. WO0198537A2, herein incorporated byreference.

Once one or more target sites have been identified, oligonucleotides arechosen that are sufficiently complementary to the target (i.e.,hybridize sufficiently well and with sufficient specificity) to give thedesired effect. For example, in preferred embodiments, antisenseoligonucleotides are targeted to or near the start codon associated withMIPOL1-ETV1 genetic rearrangements.

In the context of this invention, “hybridization,” with respect toantisense compositions and methods, means hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases. For example, adenine andthymine are complementary nucleobases that pair through the formation ofhydrogen bonds. It is understood that the sequence of an antisensecompound need not be 100% complementary to that of its target nucleicacid to be specifically hybridizable. An antisense compound isspecifically hybridizable when binding of the compound to the target DNAor RNA molecule interferes with the normal function of the target DNA orRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the antisense compoundto non-target sequences under conditions in which specific binding isdesired (i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed).

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with specificity, can be used to elucidate thefunction of particular genes. Antisense compounds are also used, forexample, to distinguish between functions of various members of abiological pathway.

The specificity and sensitivity of antisense is also applied fortherapeutic uses. For example, antisense oligonucleotides have beenemployed as therapeutic moieties in the treatment of disease states inanimals and man. Antisense oligonucleotides have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides areuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues, and animals,especially humans.

While antisense oligonucleotides are preferred, other oligomericantisense compounds, including but not limited to oligonucleotidemimetics may be used, such as are described below. Preferred antisensecompounds comprise from about 8 to about 30 nucleobases (i.e., fromabout 8 to about 30 linked bases), although both longer and shortersequences may be used. Particularly preferred antisense compounds areantisense oligonucleotides, even more preferably those comprising fromabout 12 to about 25 nucleobases.

Specific examples of preferred antisense compounds includeoligonucleotides containing modified backbones or non-naturalinternucleoside linkages. As defined herein, oligonucleotides havingmodified backbones include those that retain a phosphorus atom in thebackbone and those that do not have a phosphorus atom in the backbone.For the purposes of this specification, modified oligonucleotides thatdo not have a phosphorus atom in their internucleoside backbone can alsobe considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e., the backbone) of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Methods for preparation of PNA compounds arewell known (e.g., see U.S. Pat. Nos. 5,539,082; 5,714,331; and5,719,262, and Nielsen et al., Science 254:1497 (1991), each of which isherein incorporated by reference). Most preferred embodiments of theinvention are oligonucleotides with phosphorothioate backbones andoligonucleosides with heteroatom backbones, and in particular —CH2,—NH—O—CH2-, —CH2-N(CH3)-O—CH2- [known as a methylene (methylimino) orMMI backbone], —CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2-, and—O—N(CH3)-CH2-CH2- [wherein the native phosphodiester backbone isrepresented as —O—P—O—CH2-], amid backbone, and morpholino backbonestructures, all of which are well known (e.g., see U.S. Pat. Nos.5,489,677, 5,602,240, and 5,034,506).

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyland alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3,O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where nand m are from 1 to about 10. Other preferred oligonucleotides compriseone of the following at the 2′ position: C1 to C10 lower alkyl,substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta 78:486 [1995]) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy (i.e., aO(CH2)2ON(CH3)2 group), also known as 2′-DMAOE, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH2-O—CH2-N(CH2)2.

Other preferred modifications include 2′-methoxy(2′-O—CH3),2′-aminopropoxy(2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases are well known (e.g., see U.S. Pat.No. 3,687,808) and include other synthetic and natural nucleobases (forwhich the A, G, T, C and U abbreviations for the bases are used in thefollowing examples), such as 5-methylcytosine (5-me-C), 5-hydroxymethylC, xanthine, hypoxanthine, 2-amino-A, 6-methyl or 2-propyl and otheralkyl derivatives of A and G, 2-thio-U, 2-thio-T and 2-thio-C, 5-halo-Uand -C, 5-propynyl U and C, 6-azo U, C and T, 5-uracil (pseudouracil),4-thio-U, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted A and G, 5-halo substituted U and C, particularly 5-bromo,5-trifluoromethyl and other 5-substituted U and C, 7-methyl-G and7-methyl-A, 8-aza-G and 8-aza-A, 7-deaza-G and 7-deaza-A and 3-deaza-Gand 3-deaza-A. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyl-U and 5-propynyl-C. 5-methyl-C substitutions are known toincrease nucleic acid duplex stability and are preferred basesubstitutions in some embodiments, even more particularly when combinedwith 2′-O-methoxyethyl sugar modifications.

Another modification of the oligonucleotides involves chemically linkingto the oligonucleotide one or more moieties or conjugates that enhancethe activity, cellular distribution or cellular uptake of theoligonucleotide. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, (e.g.,hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain, (e.g.,dodecandiol or undecyl residues), a phospholipid, (e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate), a polyamine or apolyethylene glycol chain or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

One skilled in the relevant art knows well how to generateoligonucleotides containing the above-described modifications. Thepresent invention is not limited to the antisense oligonucleotidesdescribed above. Any suitable modification or substitution may be used.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. Antisense compounds may bechimeric compounds. “Chimeric” antisense compounds or “chimeras,” asused herein, are antisense compounds, particularly oligonucleotides,which contain two or more chemically distinct regions, each made up ofat least one monomer unit, i.e., a nucleotide in the case of anoligonucleotide compound. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligonucleotide mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNaseH is a cellular endonuclease thatcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of oligonucleotide inhibition of geneexpression. Consequently, comparable results can often be obtained withshorter oligonucleotides when chimeric oligonucleotides are used,compared to phosphorothioate deoxyoligonucleotides hybridizing to thesame target region. Cleavage of the RNA target can be routinely detectedby gel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds may be formed as composite structures oftwo or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.

Other embodiments include pharmaceutical compositions and formulationsthat include the antisense compounds as described herein.

B. Gene Therapy

Embodiments may use any genetic manipulation to modulate the expressionof cancer markers associated with MIPOL1-ETV1 genetic rearrangementsdescribed herein. Examples of genetic manipulation include, but are notlimited to, gene knockout (such as by removing the genetic rearrangementfrom the chromosome using, e.g., by recombination), expression ofantisense constructs with or without inducible promoters, and the like.Delivery of nucleic acid construct to cells in vitro or in vivo may beconducted using any suitable method. A suitable method is one thatintroduces the nucleic acid construct into the cell such that thedesired event occurs (e.g., expression of an antisense construct).Genetic therapy may also be used to deliver siRNA or other interferingmolecules that are expressed in vivo (e.g., upon stimulation by aninducible promoter (e.g., an androgen-responsive promoter)).

Introduction of molecules carrying genetic information into cells isachieved by any of various methods including, but not limited to,directed injection of naked DNA constructs, bombardment with goldparticles loaded with said constructs, and macromolecule mediated genetransfer using, for example, liposomes, biopolymers, and the like.Preferred methods use gene delivery vehicles derived from viruses,including, but not limited to, adenoviruses, retroviruses, vacciniaviruses, and adeno-associated viruses. Because of the higher efficiencyas compared to retroviruses, vectors derived from adenoviruses are thepreferred gene delivery vehicles for transferring nucleic acid moleculesinto host cells in vivo. Adenoviral vectors and their use in genetransfer are well known (e.g., see PCT publications WO 00/12738 and WO00/09675 and U.S. Pat. Nos. 6,033,908, 6,019,978, 6,001,557, 5,994,132,5,994,128, 5,994,106, 5,981,225, 5,885,808, 5,872,154, 5,830,730, and5,824,544, each of which is herein incorporated by reference in itsentirety). Such vectors and methods have been shown to provide veryefficient in vivo gene transfer into a variety of solid tumors in animalmodels and into human solid tumor xenografts in immune-deficient mice.

Vectors may be administered to subject in a variety of well known ways,e.g., administered into tumors or tissue associated with tumors by usingdirect injection or administration via the blood or lymphaticcirculation (See e.g., PCT publication 99/02685 herein incorporated byreference in its entirety). Exemplary dose levels of adenoviral vectorare preferably 10⁸ to 10¹¹ vector particles added to the perfusate.

C. Antibody or Small Molecule Therapies

Some embodiments are or use antibodies and/or small molecules thattarget prostate tumors that express a cancer marker associated withMIPOL1-ETV1 genetic rearrangements. In some embodiments, the therapeuticregimen is selected based on a diagnostic result and uses a suitableantibody (e.g., monoclonal, polyclonal, or synthetic) in the therapeuticmethods. In preferred embodiments, the antibodies used for cancertherapy are humanized antibodies. Methods for humanizing antibodies arewell known (See e.g., U.S. Pat. Nos. 6,180,370, 5,585,089, 6,054,297,and 5,565,332; each of which is herein incorporated by reference).

In some embodiments, the therapeutic antibodies comprise an antibodygenerated against a cancer marker associated with MIPOL1-ETV1 geneticrearrangements, wherein the antibody is conjugated to a cytotoxic agent.In such embodiments, a tumor specific therapeutic agent is generatedthat does not target normal cells, thus reducing many of the detrimentalside effects of traditional chemotherapy. For certain applications, itis envisioned that the therapeutic agents will be pharmacologic agentsthat will serve as useful agents for attachment to antibodies,particularly cytotoxic or otherwise anticellular agents having theability to kill or suppress the growth or cell division of endothelialcells. Embodiments may use any pharmacologic agent that can beconjugated to an antibody, and delivered in active form. Exemplaryanticellular agents include chemotherapeutic agents, radioisotopes, andcytotoxins. Such therapeutic antibodies may include a variety ofcytotoxic moieties, including but not limited to, radioactive isotopes(e.g., iodine-131, iodine-123, technetium-99m, indium-111, rhenium-188,rhenium-186, gallium-67, copper-67, yttrium-90, iodine-125 orastatine-211), hormones such as a steroid, antimetabolites such ascytosines (e.g., arabinoside, fluorouracil, methotrexate or aminopterin;an anthracycline; mitomycin C), vinca alkaloids (e.g., demecolcine;etoposide; mithramycin), and antitumor alkylating agent such aschlorambucil or melphalan. Other embodiments may include agents such asa coagulant, a cytokine, growth factor, bacterial endotoxin or the lipidA moiety of bacterial endotoxin. For example, in some embodiments,therapeutic agents will include plant-, fungus- or bacteria-derivedtoxin, such as an A chain toxins, a ribosome inactivating protein,α-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin orpseudomonas exotoxin, to mention just a few examples. In some preferredembodiments, deglycosylated ricin A chain is used.

In any event, it is proposed that agents such as these may, if desired,be successfully conjugated to an antibody, in a manner that will allowtheir targeting, internalization, release or presentation to bloodcomponents at the site of the targeted tumor cells as required usingknown conjugation technology (See, e.g., Ghose et al., Methods Enzymol.,93:280 [1983]).

For example, some embodiments provide immunotoxins targeted againstcancer marker associated with MIPOL1-ETV1 genetic rearrangements.Immunotoxins are conjugates of a specific targeting agent typically atumor-directed antibody or fragment, with a cytotoxic agent, such as atoxin moiety. The targeting agent directs the toxin to, and therebyselectively kills, cells carrying the targeted antigen. In someembodiments, therapeutic antibodies employ crosslinkers that providehigh in vivo stability (Thorpe et al., Cancer Res., 48:6396 [1988]).

In other embodiments, particularly those involving treatment of solidtumors, antibodies are designed to have a cytotoxic or otherwiseanticellular effect against the tumor vasculature, by suppressing thegrowth or cell division of the vascular endothelial cells. This attackis intended to lead to a tumor-localized vascular collapse, deprivingthe tumor cells, particularly those tumor cells distal of thevasculature, of oxygen and nutrients, ultimately leading to cell deathand tumor necrosis.

In preferred embodiments, antibody based therapeutics are formulated aspharmaceutical compositions as described herein. In preferredembodiments, administration of an antibody composition that targets amoiety associated with MIPOL1-ETV1 genetic rearrangements results in ameasurable decrease in cancer (e.g., decrease or elimination of tumor).

VII. Transgenic Animals

Embodiments include generation of transgenic animals comprising anexogenous cancer marker gene that is identical to or representative of aMIPOL1-ETV1 genetic rearrangement described herein, which includesmutants and variants thereof (e.g., truncations or single nucleotidepolymorphisms). In preferred embodiments, the transgenic animal displaysan altered phenotype (e.g., increased or decreased presence of markersassociated with MIPOL1-ETV1 genetic rearrangements) as compared towild-type animals. Methods for analyzing the presence or absence of suchphenotypes include but are not limited to, those disclosed herein. Insome preferred embodiments, the transgenic animals further display anincreased or decreased growth of tumors or evidence of cancer.

Such transgenic animals are useful in drug (e.g., cancer therapy)screens. In some embodiments, test compounds (e.g., a drug that issuspected of being useful to treat cancer) and control compounds (e.g.,a placebo) are administered to the transgenic animals and the controlanimals and the effects evaluated.

The transgenic animals can be generated via a variety of methods. Insome embodiments, embryonal cells at various developmental stages areused to introduce transgenes for the production of transgenic animals.Different methods are used depending on the stage of development of theembryonal cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter that allows reproducible injection of 1-2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442 [1985]). As aconsequence, all cells of the transgenic non-human animal carry theincorporated transgene. This is reflected in the efficient transmissionof the transgene to offspring of the founder since 50% of the germ cellsharbor the transgene based on standard Mendelian genetics. Methods formaking transgenics are well known (e.g., see U.S. Pat. No. 4,873,191,which is herein incorporated by reference in its entirety).

In other embodiments, retroviral infection is used to introducetransgenes into a non-human animal. In some embodiments, the retroviralvector is used to transfect oocytes by injecting the retroviral vectorinto the perivitelline space of the oocyte (U.S. Pat. No. 6,080,912,incorporated herein by reference). In other embodiments, the developingnon-human embryo can be cultured in vitro to the blastocyst stage.During this time, the blastomeres can be targets for retroviralinfection (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Hogan et al., in Manipulatingthe Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. [1986]). The viral vector system used to introduce thetransgene is typically a replication-defective retrovirus carrying thetransgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927 [1985]).Transfection is easily and efficiently obtained by culturing theblastomeres on a monolayer of virus-producing cells (Stewart, et al.,EMBO J., 6:383 [1987]). Alternatively, infection can be performed at alater stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner et al., Nature 298:623 [1982]). Most of the founderswill be mosaic for the transgene since incorporation occurs only in asubset of cells that form the transgenic animal. Further, the foundermay contain various retroviral insertions of the transgene at differentpositions in the genome that generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into thegermline, albeit with low efficiency, by intrauterine retroviralinfection of the midgestation embryo (Jahner et al., supra [1982]).Additional means of using retroviruses or retroviral vectors to createtransgenic animals known to the art involve the micro-injection ofretroviral particles or mitomycin C-treated cells producing retrovirusinto the perivitelline space of fertilized eggs or early embryos (PCTInternational Application WO 90/08832 [1990], and Haskell and Bowen,Mol. Reprod. Dev., 40:386 [1995]).

In other embodiments, the transgene is introduced into embryonic stemcells and the transfected stem cells are used to form an embryo. EScells are obtained by culturing pre-implantation embryos in vitro underappropriate conditions (Evans et al., Nature 292:154 [1981]; Bradley etal., Nature 309:255 [1984]; Gossler et al., Proc. Acad. Sci. USA 83:9065[1986]; and Robertson et al., Nature 322:445 [1986]). Transgenes can beefficiently introduced into the ES cells by DNA transfection by avariety of methods known to the art including calcium phosphateco-precipitation, protoplast or spheroplast fusion, lipofection andDEAE-dextran-mediated transfection. Transgenes may also be introducedinto ES cells by retrovirus-mediated transduction or by micro-injection.Such transfected ES cells can thereafter colonize an embryo followingtheir introduction into the blastocoel of a blastocyst-stage embryo andcontribute to the germ line of the resulting chimeric animal (forreview, See, Jaenisch, Science 240:1468 [1988]). Prior to theintroduction of transfected ES cells into the blastocoel, thetransfected ES cells may be subjected to various selection protocols toenrich for ES cells which have integrated the transgene assuming thatthe transgene provides a means for such selection. Alternatively, thepolymerase chain reaction may be used to screen for ES cells that haveintegrated the transgene. This technique obviates the need for growth ofthe transfected ES cells under appropriate selective conditions prior totransfer into the blastocoel.

In still other embodiments, homologous recombination is used toknock-out gene function or create deletion mutants (e.g., truncationmutants), using well known methods (see U.S. Pat. No. 5,614,396,incorporated herein by reference).

Experimental

The following examples are provided to demonstrate and illustratecertain preferred embodiments and aspects of the compositions andmethods disclosed herein, but are not to be construed as limiting thescope of the claimed invention.

EXAMPLE 1 ETV1 Generic Rearrangements in Cell Lines

This example shows that LNCaP and MDA-PCa 2B cell lines harborrearrangements that localize the ETV1 locus to 14q13.3-14q21.1.

Experiments were designed to identify cell line models of aberrant ETV1activation. The prostate cancer cell line LNCaP has markedlyover-expressed ETV1, but RLM-RACE revealed expression of only the wildtype transcript (Tomlins et al., Science 310, 644-8 (2005)). Theseexperiments were designed to identify in LNCaP cells a novelrearrangement affecting the expression of ETV1. Thus, a split-probe FISHstrategy, consisting of one probe on and one probe 5′ to the ETV1 locus,was used to look for gross rearrangements involving ETV1 (FIG. 1).

Interphase FISH on formalin-fixed paraffin-embedded (FFPE) tissuesections was performed using known methods (substantially as previouslydescribed in Tomlins et al., Cancer Res 66, 3396-400 (2006)). A minimumof 50 nuclei per assay were evaluated. Metaphase spreads of LNCaP andMDA-PCa 2B were prepared using standard cytogenetic techniques. Slideswere pre-treated in 2×SSC for 2 min, 70% ethanol for 2 min and 100%ethanol for 2 min, and air dried. Slide samples and probes wereco-denatured at 75° C. for 2 min, and hybridized overnight at 37° C.Post-hybridization was in 0.5×SSC at 42° C. for 5 min, followed by 3washes in PBST. Fluorescent detection was performed usinganti-digoxigenin conjugated to fluorescein (Roche Applied Science,Indianapolis, Ind.) and streptavidin conjugated to a fluorophore (AlexaFluor 594, Invitrogen). Slides were counterstained and mounted usingstandard reagents and methods (in ProLong Gold Antifade Reagent withDAPI, Invitrogen). Slides were examined using a fluorescence microscope(Axio Imager Z1, Zeiss, Thornwood, N.Y.) and imaged with a CCD camerausing a standard algorithm to analyze results (ISIS software,Metasystems, Altlussheim, Germany). BACs (listed in Table 2, obtainedfrom the BACPAC Resource Center (Oakland, Calif.)) were used to prepareprobes using previously described methods (Tomline et al., 2006, supra).Pre-labeled chromosome 7 centromere and 7p telomeric probes were alsoused (from Vysis Corp., Des Plaines, Ill.). The integrity and correctlocalization of all probes were verified by hybridization to metaphasespreads of normal peripheral lymphocytes.

On LNCaP metaphases, this assay revealed two pairs of co-localizingsignals at the ETV1 locus on 7p, and two split signals where the 5′signals remained on 7p, while two copies of the ETV1 locus were insertedinto another chromosome (FIG. 1c ). This rearrangement in 2 of 4 copiesof chromosome 7 is consistent with other rearrangements observed byG-banding or spectral karyotyping (SKY) in tetraploid LNCap cells(Beheshti et al., Mol Diagn 5, 23-32 (2000); Beheshti et al., Neoplasia3, 62-9 (2001); Gibas et al., Cancer Genet Cytogenet 11, 399-404 (1984);van Bokhoven et al., Prostate 57, 226-44 (2003)). Cytogenetic analysisindicated that ETV1 was inserted into chromosome 14, and this wasconfirmed by using a FISH mapped BAC previously localized to 14q (FIG.2). Subsequent FISH assays were used to determine that the break onchromosome 7 was localized completely 5′ to the ETV1 locus, consistentwith RACE revealing over-expression of full length ETV1 (FIG. 3).

To localize the breakpoint, Southern blotting was performed using 22probes across the implicated region of 7p. Genomic DNA (10 μg) wasdigested with EcoRI or PstI (New England Biologicals, Ipswich, Mass.)overnight. Fragments were resolved on a 0.8% agarose gel at 40 Vovernight, transferred to a nylon membrane (Hybond NX), prehybridized,hybridized with probe and washed according to standard laboratoryprocedures. A series of 22 probes spanning the region of chr 7implicated by FISH (between RP11-313C20 and RP11-703A4) were generatedby PCR amplification using pooled normal human male genomic DNA astemplate, a high fidelity polymerase enzyme (Platinum Taq HighFidelity), and primers listed in Table 1. Twenty-five ng of each probewas labeled with dCTP-P32 and used for hybridization.

A single probe (Probe A, see Table 1, “Southern Probe”) showed evidenceof a rearrangement, with both EcoRI and PstI digested LNCaP DNA showingadditional bands (FIG. 3).

PstI digested DNA was then used for inverse PCR to identify the genomicbreakpoint sequence on chromosome 7 as well as the partner sequence(FIG. 4). Primers A1, A2, A3, which are reverse complemented from thewildtype sequence and are divergent to primers B1, B2, B3, were used forinverse PCR on PstI digested DNA and religated (intramolecular ligation)using the LNCaP genomic DNA template. Nested PCRs were performed in thefollowing order of primer combinations: A1 and B1, then A2 and B2, andfinally A3 and B3. The fusion product was amplified using PCRamplification and standard laboratory procedures (Expand 20 kbplus PCRSystem, Roche Diagnostics GmbH, Mannheim, Germany, used according to themanufacturor's instructions). The enriched 3 Kb band observed in nestedPCRs was cloned into a vector (pCR8/GW/TOPO, Invitrogen), and DNAisolated from various clones were screened for inserts and positiveclones were sequenced using standard laboratory procedures (by theUniversity of Michigan DNA Sequencing Core Laboratory, Ann Arbor,Mich.). Clones of the fusions sequences were confirmed by PCRamplification (Platinum Taq High Fidelity system) using fusion specificprimers (genomic fusion f and r; Table 1).

Sequencing of the amplified product confirmed the breakpoint onchromosome 7p located in a region identified by using Southern blotting,and the insertion point was an intronic sequence from the MIPOL1 locusat chromosome 14q13.3-14q21.1 (FIGS. 1b and 4), consistent with theresults obtained by FISH analysis described above. This rearrangementwas confirmed by PCR amplification of a product made by using undigestedLNCaP genomic DNA as template, isolated from two different passages,while no product was amplified from VCaP, normal male, or normalplacental genomic DNA (FIG. 4). Consistent with cytogenetic and SKY datarevealing no gross rearrangements in chromosomes 7 or 14 in LNCaPcells29-32, FISH analysis using split probes around the 14q13.3-14q21.1locus showed co-localized signals (FIG. 1c ). These results areconsistent with the interpretation that a limited insertion occurredaround ETV1, or the entire 7p arm telomeric to ETV1 (approximately 14MB) was deleted. The latter possibility was ruled out because highdensity aCGH did not reveal the presence of any deletions telomeric toETV1, and FISH demonstrated intact 7p telomeric sequence on all copiesof chromosome 7 (FIG. 5). Therefore, these results confirm a crypticinsertion of a minimal region around ETV1 into 14q13.3-14q21.1 in LNCaP.

Additional prostate cancer cell lines were tested to screen for ERG andETV1 expression by quantitative PCR (qPCR), to identify additionalgenetic rearrangements. The following cells were tested: an immortalizedbenign prostate epithelial cell line (RWPE) and 8 prostate cancer celllines (MDA-PCa 2B, LNCaP, VCaP, LAPC4, 22Rv1, NCI-H660, PC3 and DU145).Quantitative PCR (QPCR) was performed by using the oligonucleotideprimers shown in Table 1 (“Fusion QPCR” and “Androgen QPCR” sections)with standard laboratory procedures and reagents (Power SYBR GreenMastermix and 7300 Real Time PCR system, Applied Biosystems, FosterCity, Calif.). All oligonucleotide primers were synthesized by standardmethods (performed by Integrated DNA Technologies, Coralville, Iowa).HMBS and GAPDHS, and PSA6 primers were as described previously(Vandesompele et al., Genome Biol. 3:RESEARCH0034 (2002); Specht et al.,Am J. Pathol. 158: 419 (2001)). Androgen stimulation reactions wereperformed in quadruplicate, and all other reactions were performed induplicate.

Marked over-expression of ETV1 was detected in LNCaP3 cells, andover-expression of ERG was detected in VCaP and NCI-H660 cells, whichare both TMPRSS2:ERG positive (Tomlins et al., Science 310, 644-8(2005)). Results showed that MDA-PCa 2B cells expressed higher levels ofETV1 than LNCaP cells (FIG. 7).

MDA-PCa 2B cells were also analyzed for genetic rearrangement involvingthe ETV1 locus. Analysis by RLM-RACE revealed only full-length ETV1 madein MDA-PCa 2B cells, indicating that rearrangement involves the entireETV1 locus. A previous SKY and G-banding analysis of DNA from MDA-PCa 2Bcells demonstrated the presence of a balanced t(7;14)(p21;q21)32, thelocations of the ETV1 and MIPOL1 loci. The same FISH analysis wasperformed for MDA-PCa 2B cells as described above for LNCaP cells, usingsplit probes around the ETV1 locus and the 14q13.3-14q21.1 regions. Theresults demonstrated that MDA-PCa 2B cells also harbor a rearrangementinvolving ETV1, in which the whole ETV1 locus is translocated to the d14chromosome (FIG. 1d ). FISH revealed that the 1.5 MB 14q13.3-14q21.1region is the partner of this balanced translocation, as the telomeric14q13.3-14q21.1 probe is translocated to the d7 chromosome (FIG. 1d ).

EXAMPLE 2 Aberrant Expression of ETV1 Associated with GeneticRearrangements

This example shows that region 14q13.3-14q21.1 is coordinately regulatedin prostate cancer and LNCaP cells.

The existence of mechanistically distinct rearrangements resulting inthe localization of ETV1 to 14q13.3-14q21.1 (FIG. 1, e-g) in prostatecancer cell lines with outlier expression of ETV1 indicates thatelements at this region mediate aberrant ETV1 transcription. Thestructure of the genetic rearrangement in LNCaP cells indicates thatETV1 does not acquire a new proximal promoter after insertion into the14q13.3-14q21.1 locus because approximately 200kb of the 5′ upstreamsequence is inserted along with the ETV1 locus (see FIG. 1a ).Furthermore, while expression of MIPOL1 in the prostate has not beendescribed, FOXA1, immediately adjacent to MIPOL1, is strongly expressedthroughout human and murine prostate development and acts as atranscriptional potentiator for androgen-regulated gene expression(Mirosevich et al., Prostate 66, 1013-28 (2006); Mirosevich et al.,Prostate 62, 339-52 (2005)). Over-expression of ETV1 in these lines maybe driven by enhancer elements that confer coordinated prostatespecificity or androgen regulation to this region. To test thishypothesis, the expression of four contiguous transcripts in the 1.5 MB14q13.3-14q21.1 region (SLC25A21, MIPOL1, FOXA1 and TTC6) was assessedusing data in the expO dataset (International Genomics Consortium (IGC)expression project for Oncology (expO)). The expO dataset is amulti-tumor gene expression dataset generated by a consortium of labsincluding TGEN and has been included in Oncomine (Rhodes et al.,Neoplasia 6, 1-6 (2004)). All four transcripts showed significantover-expression in prostate cancer compared to all other cancers (FIG.6a ). Furthermore, when all measured genes on chromosome 14 were rankedby significant over-expression in prostate cancer compared to all othercancers in this data set, MIPOL1 ranked second, FOXA1 ranked fifth, andTTC6 ranked twenty-third.

Although all four transcripts were over-expressed in prostate cancer,stimulation of LNCaP with R1881 minimally increased the expression ofFOXA1 (1.3 fold, p=0.05), while SLC25A21 (0.75 fold, p=0.008), MIPOL1(1.2 fold, p=0.08) and TTC6 (0.70 fold, p=0.10) showed no significantchange or reduced expression following R1881 stimulation (FIG. 6b ).However, stimulation of LNCaP with R1881 resulted in significantlyincreased expression of ETV1 (1.8 fold, p=0.0004, FIG. 6c ), indicatingthat rearrangement to this region confers androgen-responsiveness toETV1, in addition to aberrant expression. Together, these results showcoordinated over-expression in prostate cancer for the genes in thisregion (and ETV1 when inserted therein), with additional regulationmediated by androgen.

To model progression to hormone refractory metastatic disease, LNCaPcells have previously been cultured in the absence of androgen andclones were selected that are insensitive to androgen. These modelsystems thus are related to expression levels of genetic components andandrogen regulated expression of genetic components in the cells, whichwould include ETV1 and genes at the 14q13.3-14q21.1 region contained inthe LNCaP cell line and the derived clones made from this cell line. Ourinvestigation of all of these independently derived cell lines for whichpublicly available expression profiling data is available, ETV1 showsmarked down-regulation in androgen insensitive derivatives of LNCaP36-39cells. To confirm these findings, gene expression in LNCaP and itsandrogen insensitive derivative C4-2B40 were profiled by using ahybridization based assay to examine gene expression (Agilent WholeGenome Microarrays). As shown in FIG. 6d , ETV1 was the fourth mostover-expressed feature in LNCaP compared to C4-2B (307-fold difference).When measured by using qPCR, the marked down-regulation of ETV1 in C4-2B(about 22,500 fold), compared to the parental LNCaP cell line wasconfirmed (FIG. 6e ). Furthermore, qPCR also demonstrated that C4-2Bcells express less PSA (3-fold less) and the four genes at14q13.3-14q21.1, when compared to LNCaP cells (FIG. 6, f-g). Theseresults demonstrate coordinated regulation of androgen induced genes andgenes at 14q13.3-14q21.1. These results indicate that ETV1 is markedlydown-regulated in androgen insensitive LNCaP derivatives either due todeletion or transcriptional mechanisms, or clones that do not harbor theins(7;14) are selected by using the androgen deprivation method used toderive these clones from the LNCaP cell line. By using FISH and PCRanalysis on genomic DNA, the data confirmed that C4-2B cells harbor thesame ETV1 rearrangement as LNCaP cells (FIG. 8), leading to theconclusion that the down-regulation of ETV1 is due to mutation ortranscriptional changes in the derivative clones.

TABLE 1 Oligo- Bases nucleotide SEQ Gene/ Sequence Within PrimerSequence  ID Assay Region (Accession No.) Sequence Name (5′ to 3′) NOAndrogen/ ETV1 NM_004956.3  624-645 ETV1_exon_6-7_ CTACCCCATGGACCACAG  1Expression f ATTT QPCR Androgen/ ETV1 NM_004956.3  771-750ETV1_exon_6-7_ CTTAAAGCCTTGTGGTGG  2 Expression r GAAG QPCR ExpressionERG NM_004449.3  574-597 ERG_exon_5-6_f CGCAGAGTTATCGTGCCA  3 QPCRGCAGAT Expression ERG NM_004449.3  659-636 ERG_exon_5-6rCCATATTCTTTCACCGCC  4 QPCR CACTCC RLM-RACE NA NA Generacer 5′_fCGACTGGAGCACGAGGA  5 CACTGA RLM-RACE ETV1 NM_004449.3  374-351ETV1_exon 4-5_r CATGGACTGTGGGGTTCT  6 TTCTTG RLM-RACE ETV1 NM_004449.3 735-710 ETV1_exon 7-r AGACATCTGGCGTTGGTA  7 CATAGGAC Fusion QPCR HERV-BC020811.1  303-327 HERV-K:ETV1f GAGTCCCAAGTACGTCCA  8 K_22q11.23CGGTCAG Fusion QPCR ETV1 NM_004956.3  371-345 HERV-K:ETV1-rGGACTGTGGGGTTCTTTC  9 TTGATTTTC Fusion QPCR HNRPA2B1 NM_002137.2 136-155 HNRPA2B1:ETV1- TGCGGGAAATCGGGCTGA 10 f AG Fusion QPCR ETV1NM_004956.3  181-154 HNRPA2B1:ETV1- TTTTCCTGACATTTGTTGG 11 r TTTCTCGTTFusion QPCR SLC45A3 NM_033102.2   74-92 SLC45A3:ETV1-fCGCTGGCTCCGGGTGACA 12 G Fusion QPCR ETV1 NM_004956.3  366-340SLC45A3:ETV1-r GTGGGGTTCTTTCTTGATT 13 TTCAGTGG Fusion QPCR C15ORF21NM_001005266.1  313-336 C15ORF21:ETV1-f CAACTAACACTGCGGCTT 14 CCTGAGFusion QPCR ETV1 NM_004956.3  483-461 C15ORF21:ETV1-r CATTCCCACTTGTGGCTT15 CTGAT Androgen QPCR TMPRSS2 NM_005656.2 1539-1563 TMPRSS2-fCAGGAGTGTACGGGAAT 16 GTGATGGT Androgen QPCR TMPRSS2 NM_005656.21608-1585 TMPRSS2-r GATTAGCCGTCTGCCCTC 17 ATTTGT Androgen QPCR TTC6NM_001007795.1 1080-1108 TTC6-f TGCCATGAAGATCAGTAC 18 TACAGCAGAATAndrogen QPCR TTC6 NM_001007795.1 1150-1125 TTC6-r GTGGCCCATAAACTCATG 19AATCACC Androgen QPCR SLC25A21 NM_030631.1  356-377 SLC25A21-fCAGATCGTGGCCGGTGGT 20 TCT Androgen QPCR SLC25A21 NM_030631.1  408-483SLC25A21-r GGGTGCATCAGGCAAATT 21 TCTACAAG Androgen QPCR MIPOL1NM_138731.2 1607-1633 MIPOL1-f CAACAACAAAATGAGGA 22 ACTGGCTACTAndrogen QPCR MIPOL1 NM_138731.2 1673-1649 MIPOL1-r ATTCCATATTTGCTCGCTC23 TGTCAG Androgen QPCR FOXA1 NM_004496.2  327-350 FOXA1-fGAAGATGGAAGGGCATG 24 AAACCAG Androgen QPCR FOXA1 NM_004496.2  408-389FOXA1-r GCTGACCGGGACGGAGG 25 AGT Androgen QPCR SLC45A3 NM_033102.21223-1242 SLC45A3-f TCGTGGGCGAGGGGCTGT 26 A Androgen QPCR SLC45A3NM_033102.2 1308-1284 SLC45A3-r CATCCGAACGCCTTCATC 27 ATAGTGTAndrogen QPCR HERV- BC020811.1  168-194 HERV- CTTTTCTCTAGGGTGAAG 28K_22q11.23 K_22q11.23-f GGACTCTCG Androgen QPCR HERV- BC020811.1 263-238 HERV- CTTCACCCACAAGGCTCA 29 K_22q11.23 K_22q11.23-r CTGTAGACAndrogen QPCR HNRPA2B1 NM_002137.2  594-620 HNRPA2B1-fGCTTTGGCTTTGTTACTTT 30 TGATGACC Androgen QPCR HNRPA2B1 NM_002137.2 693-665 HNRPA2B1-r GCCTTTCTTACTTCTGCAT 31 TATGACCATT Androgen QPCRC15ORF21 NM_001005266.1  219-243 C15ORF21-f AAGGACGTGCAAGGATG 32TTTTTATT Androgen QPCR C15ORF21 NM_001005266.1  293-274 C15ORF21-rATGGGAAGATGGGGGCT 33 GTT Southern probe Chr 7 (5 to NT_007819.1613,685,335- LNCAP_A-f GTCAATGGCTAAAAGATG 34 ETV1) 13,685,364GATAAAAGTGGA Southern probe Chr 7 (5′ to NT_007819.16 13,686,833-LNCAP_A-r CAGATAGAAGAGGGGTT 35 ETV1) 13,686,804 AGCAAAATGTGTTSouthern probe Chr 7 (5′ to NT_007819.16 13,690,754- LNCAP_B1-fCAGAAGGCAAATGTGAG 36 ETV1) 13,690,779 AGGATAGTC Southern probe Chr 7 (5′to NT_007819.16 13,691,679- LNCAP_B1-r CTGGATCTGTAACACCCG 37 ETV1)13,691,657 TGAGC Southern probe Chr 7 (5′ to NT_007819.16 13,693,600-LNCAP_B2-f AAAAAGCAAAGACAAGA 38 ETV1) 13,693,625 CCGTGGATTSouthern probe Chr 7 (5′ to NT_007819.16 13,695,054- LNCAP_B2-rGAACTACCTGCGTGCTGA 39 ETV1) 13,695,028 CTTGGAGAT Southern probeChr 7 (5′ to NT_007819.16 13,699,772- LNCAP_C-f AAAAGGCAAAGAGGGGT 40ETV1) 13,699,801 TAAAACATACATA Southern probe Chr 7 (5′ to NT_007819.1613,700,641- LNCAP_C-r AACCCCTCCTTCCACTTCT 41 ETV1) 13,700,618 CCACTSouthern probe Chr 7 (5′ to NT_007819.16 13,705,005- LNCAP_D-fTGGAGGCATAGAAAAGC 42 ETV1) 13,705,034 TGAGAAATAAG Southern probeChr 7 (5′ to NT_007819.16 13,705,856- LNCAP_D-r TTGGTGCTAGAAGAACTG 43ETV1) 13,705,830 GGAGAAAC Southern probe Chr 7 (5′ to NT_007819.1613,711,263- LNCAP_F-f GAAAGTCAGGGGCACAT 44 ETV1) 13,711,291 ATAGATTAGAGSouthern probe Chr 7 (5′ to NT_007819.16 13,712,112- LNCAP_F-rGCCTTCCCCATACAGTTT 45 ETV1) 13,712,089 CTCCTT Southern probe Chr 7 (5′to NT_007819.16 13,708,247- LNCAP_E-f AAGTTCGTTAAGCCCAGG 46 ETV1)13,708,275 ATCGTAGGTA Southern probe Chr 7 (5′ to NT_007819.1613,709,539- LNCAP_E-r ATATGAAGCCAGCAGCCA 47 ETV1) 13,709,514 GGTAGCASouthern probe Chr 7 (5′ to NT_007819.16 13,719,560- LNCAP_G-fTTAGATAAACTGAAAGCC 48 ETV1) 13,719,587 GAACCTGAAC Southern probeChr 7 (5′ to NT_007819.16 13,720,465- LNCAP_G-r CAAACTGGCAAGCAATGT 49ETV1) 13,720,441 GAACTGT Southern probe Chr 7 (5′ to NT_007819.1613,723,583- LNCAP_H-f TCACCGACAAAACCCATA 50 ETV1) 13,723,615 GAGAAAGAGTSouthern probe Chr 7 (5′ to NT_007819.16 13,724,850- LNCAP_H-rTTAAATGGTGAGGCAATG 51 ETV1) 13,724,823 AGGAAAGTG Southern probeChr 7 (5′ to NT_007819.16 13,727,707- LNCAP_I-f TTGCTCATTCTCTTTCTCC 52ETV1) 13,727,735 CCTACACTAA Southern probe Chr 7 (5′ to NT_007819.1613,729,407- LNCAP_I-r TCCCCACCACCAACCATC 53 ETV1) 13,729,387 CTCSouthern probe Chr 7 (5′ to NT_007819.16 13,730,208- LNCAP_J1-fCTGGGGGAAAAGCAAGT 54 ETV1) 13,730,233 AGGAAAGTA Southern probe Chr 7 (5′to NT_007819.16 13,731,073- LNCAP_J1-r ACAAGAGTTAGTCACGGC 55 ETV1)13,731,047 AAAGGAGTT Southern probe Chr 7 (5′ to NT_007819.1613,733,199- LNCAP_J2-f GCCCTTTGCCCATGAGAA 56 ETV1) 13,733,220 CTAASouthern probe Chr 7(5 to NT_007819.16 13,734,028- LNCAP_J2-rTCCCAGAAGAGATGATAT 57 ETV1) 13,734,003 GAGGTGTC Southern probe Chr 7 (5′to NT_007819.16 13,737,858- LNCAP_K-f TCAGTCCCATCTCCCCCT 58 ETV1)13,737,881 AAACCA Southern probe Chr 7 (5′ to NT_007819.16 13,739,203-LNCAP_K-r CACCATTCTCACCCGACC 59 ETV1) 13,739,180 ACATTG Southern probeChr 7(5′ to NT_007819.16 13,743,031- LNCAP_L-f TGTAAACTGCAATGAAAA 60ETV1) 13,743,060 GAAAAGAAAAAG Southern probe Chr 7(5′ to NT_007819.1613,744,016- LNCAP_L-r CAAGAGATGGGAGAGGA 61 ETV1) 13,743,987AGAATGAATAATA Southern probe Chr 7 (5′ to NT_007819.16 13,746,316-LNCAP_M-f CTATCTAGTCCCTTACGC 62 ETV1) 13,746,343 TTTCCCTGTGSouthern probe Chr 7 (5′ to NT_007819.16 13,747,138- LNCAP_M-rCATTAGCATTTGGCCTTT 63 ETV1) 13,747,116 GGTCA Southern probe Chr 7 (5′ toNT_007819.16 13,754,560- LNCAP_N1-f TGCCTCCCCATAAGTCAC 64 ETV1)13,754,584 CAATCTC Southern probe Chr 7 (5′ to NT_007819.16 13,755,734-LNCAP_N1-r CCTGTATTCTAACCCTGG 65 ETV1) 13,755,705 ACTTCTCATCAASouthern probe Chr 7 (5′ to NT_007819.16 13,756,219- LNCAP_N2-fCTTGTTTATTGGCCTAGTC 66 ETV1) 13,756,246 CTTTGTGCT Southern probeChr 7 (5′ to NT_007819.16 13,757,204- LNCAP_N2-r GCTTTGTGGGTAGTCCTG 67ETV1) 13,757,179 TCTGAGTG Southern probe Chr 7 (5′ to NT_007819.1613,760,623- LNCAP_O1-f GGCCCATCCCGGTTTGCT 68 ETV1) 13,760,642 AASouthern probe Chr 7 (5′ to NT_007819.16 13,761,824- LNCAP_O1-rGTTTCCCCACCACTTCCTT 69 ETV1) 13,761,798 TCTATGTC Southern probeChr 7 (5′ to NT_007819.16 13,764,545- LNCAP_O2-f GCACAAGACATACACGC 70ETV1) 13,764,569 AGATACAC Southern probe Chr 7 (5′ to NT_007819.1613,765,703- LNCAP_O2-r AACGCTGGACTATGGAAC 71 ETV1) 13,765,678 TTTACCTGSouthern probe Chr 7 (5′ to NT_007819.16 13,767,646- LNCAP_P1-fTCCTCTCATTCATTTTGCA 72 ETV1) 13,767,674 TTCGTGTTAG Southern probeChr 7 (5′ to NT_007819.16 13,768,845- LNCAP_P1-r GGCTTTGAGGGATTACTG 73ETV1) 3213,768,819 GGTTGTTCT Southern probe Chr 7(5′ to NT_007819.1613,770,885- LNCAP_P2-f GCAGGGCAAAGAAGCAG 74 ETV1) 13,770,905 TAGGSouthern probe Chr 7 (5′ to NT_007819.16 13,772,453- LNCAP_P2-rGGATCCCAATTTAGTTTC 75 ETV1) 13,772,428 AAGTTACG Southern probe Chr 7 (5′to NT_007819.16 13,774,561- LNCAP_Q-f ATGTGCTGGCTAGATTGG 76 ETV1)13,774,586 ACTGAAAA Southern probe Chr 7(5′ to NT_007819.16 13,775,272-LNCAP_Q-r CAATAAAGCTGGAGGGG 77 ETV1) 13,775,244 TGATAAATAAAT Inverse PCRChr 7 (Probe NT_007819.16 13,685,817- Inverse A1 TTAGAAGGAGACAATCTT 78A) 13,685,793 ATTCCAG Inverse PCR Chr 7 (Probe NT_007819.16  13685,657-Inverse A2 CTCTTAAAGAGATGAAGC 79 A) 13,685,634 AGGGAG Inverse PCRChr 7 (Probe NT_007819.16 13,685,626- Inverse A3 TTGGCTAGATACAGGGTG 80A) 13,685,603 AATATT Inverse PCR Chr 7 (Probe NT_007819.16 13,685,833-Inverse B1 TGAATTCATGTGTGTAGC 81 A) 13,685,856 TGAGCC Inverse PCRChr 7 (Probe NT_007819.16 13,685,860- Inverse B2 TGACAGCGGGAATAAAG 82 A)13,685,883 TACATGC Inverse PCR Chr 7 (Probe NT_007819.16 13,686,095-Inverse B3 GTTGGGAGGTTTACTTGC 83 A) 13,686,118 CAATTA Genomic fusionChr 7 (5′ to NT_007819.16 13,686,807- Genomic fusion- ACATTTTGCTAACCCCTC84 ETV1) 13,686,832 f TTCTATCT Genomic fusion Chr 14 NT_026437.1118,985,248- Genomic fusion- TCAACCTCAAAAATAAAT 85 (MIPOL1) 18,985,272 rGGCATCT RWPE-ETV1 SERPINE1 NM_000602.1 1181-1200 SERPINE1-fGCATGGCCCCCGAGGAG 86 AT RWPE-ETV1 SERPINE1 NM_000602.1 1270-1248SERPINE1-r CTTGGCCCATGAAAAGGA 87 CTGTT RWPE-ETV1 TGFBI NM_000358.11506-1528 TGFBI-f AGGTACGGGACCCTGTTC 88 ACGAT RWPE-ETV1 TGFBINM_000358.1 1605-1580 TGFBI-r CTACCAGCATGCTAAAGC 89 GATTGTCT RWPE-ETV1IGFBP3 NM_000598.4  738-762 IGFBP3-f CGAGTCCAAGCGGGAGA 90 CAGAATARWPE-ETV1 IGFBP3 NM_000598.4  837-814 IGFBP3-r TACACCCCTGGGACTCAG 91CACATT RWPE-ETV1 MMP3 NM_002422.3 1055-1080 MMP3-f TTCATTTTGGCCATCTCTT92 CCTTCAG RWPE-ETV1 MMP3 NM_002422.3 1181-1155 MMP3-rTATCCAGCTCGTACCTCA 93 TTTCCTCT RWPE-ETV1 SPOCK1 NM_004598.3  829-848SPOCK1-f GCCCACCAGCTCCAACAC 94 AG RWPE-ETV1 SPOCK1 NM_004598.3  951-928SPOCK1-r GAAGGGTCAAGCAGGAG 95 GTCATAG RWPE-ETV1 BCL2 NM_000633.21014-1039 BCL2-f CCCTGTGGATGACTGAGT 96 ACCTGAAC RWPE-ETV1 BCL2NM_000633.2 1084-1064 BCL2-r GGCATCCCAGCCTCCGTT 97 ATC RWPE-ETV1 MMP14NM_004995.2 1036-1059 MMP14-f AATTTTGTGCTGCCCGAT 98 GATGAC RWPE-ETV1MMP14 NM_004995.2 1151-1129 MMP14-r GGAACAGAAGGCCGGGA 99 GGTAGTRWPE-ETV1 MMP2 NM_004530.2  953-974 MMP2-f GAAGGCCAAGTGGTCCGT 100 GTGARWPE-ETV1 MMP2 NM_004530.2 1044-1019 MMP2-r CAGCTGTTGTACTCCTTG 101CCATTGAA RWPE-ETV1 ADAM19 NM_023038.3 2146-2165 ADAM19-fGCCTATGCCCCCTGAGAG 102 TG RWPE-ETV1 ADAM19 NM_023038.3 2271-2245ADAM19-r GCTTGAGTTGGCCTAGTT 103 TGTTGTTC RWPE-ETV1 MMP9 NM_004994.21181-1201 MMP9-f TGCCCGGACCAAGGATAC 104 AGT RWPE-ETV1 MMP9 NM_004994.21239-1221 MMP9-r AGCGCGTGGCCGAACTCA 105 T RWPE-ETV1 PLAU NM_002658.21169-1194 PLAU-f TACGGCTCTGAAGTCACC 106 ACCAAAAT RWPE-ETV1 PLAUNM_002658.2 1308-1286 PLAU-r CCCCAGCTCACAATTCCA 107 GTCAA

TABLE 2 Probe # Gene/Region Localization Probe 1 ETV1 3′ RP11-124L22 2ETV1 5′ RP11-703A4 3 Chr 14q13.3-14q21.1 C RP11-945C4 4 Chr14q13.3-14q21.1 T RP11-107E23 5 HNRPA2B1 3′ 3′ RP11-11F13 6 HNRPA2B1 5′5′ RP11-379M24 7 HERV-K_22q11.23 5′ 5′ RP11-61N10 8 HERV-K_22q11.23 3′3′ RP11-71G19 9 SLC45A3 3′ RP11-249h15 10 SLC45A3 5′ RP11-131E5 11C15ORF21 5′ RP11-474E1 12 C15ORF21 3′ RP11-626F7 13 14q32 3′ RP11-483K1314 ETV1 5′ RP11-313C20 15 Chr 7 centromere C CEP 7 16 Chr 7p telomere TTelVysion 7p 17 TMPRSS2 5′ RP11-35C4

All publications, patents, patent applications and sequences identifiedby accession numbers mentioned in the above specification are hereinincorporated by reference in their entirety. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Modifications and variations of the describedcompositions and methods of the invention that do not significantlychange the functional features of the compositions and methods describedherein are intended to be within the scope of the following claims.

1-10. (canceled)
 11. A method for detecting a MIPOL1-ETV1 geneticrearrangement and one or more markers associated with prostate cancer ina biological sample, the method comprising: a) contacting a biologicalsample with: i) a probe directly labeled with a detectable label andcomprising a sequence that is complementary to a junction at which anETV1 gene is inserted into a MIPOL1 gene; ii) a first probe directlylabeled with a detectable label and comprising a sequence complementaryto a MIPOL1 gene and a second probe directly labeled with a detectablelabel and comprising a sequence complementary to an ETV1 gene; or c) afirst amplification oligonucleotide comprising a sequence complementaryto a MIPOL1 gene, a second amplification oligonucleotide comprising asequence complementary to an ETV1 gene, and a probe directly labeledwith a detectable label and comprising a sequence complementary to theproduct produced from the first and second primers; and b) detecting inthe biological sample expression of a marker selected from the set ofmarkers consisting of AMACR/P504S, PCA3, PCGEM1, prostein/P501S, P503S,P504S, P509S, P510S, prostase/P703P, P710P, prostate specific antigen(PSA), prostatic acid phosphatase (PAP), prostate binding protein (PBP),ABCC5(MDR5), ADAMTS1, AMACR, ANNEXINA11, ANNEXINA1, ANNEXINA4, APP,ARHB, ASNS, ATF2, C1S, C4BPA, C7, CATHEPSINB, CATHEPSINH, CAVEOLIN2,CCND2, CFLAR, CLUSTERIN, COL15A1, COL1A2, COL3A1, c-terminal bindingprotein, CTBP1, CTBP2, CYSTATINC, E2EPF, EDNRB, EGR1, EPHA1, ETS2, EZH2,FASN, FAT, FHL1, FIBRONECTIN1, FKBP5, FLS353, FOLH1, FOSB, FZD7,GELSOLIN, GP73, GSTM1, GSTM3, GSTM5, GSTP1, HEPSIN, HEVIN, IGFBP3,IGFBP5, IL1R1, IL1R2, ITGA1, ITGB4, ITM2C, JUN, KERATIN5, LIMK1,LUMICAN, MADH4, MAP3K10, MAPK6, MCAM, MEIS1, MEIS2, MMECD10, MOESIN,MPDZ, MTA1, MYBL2, MYLK, NBL1, NCK1, NRAS, PCM1, pim-1, PLA2G2A, PP1CB,PPP2CB, PRKCL2, PSG9, RAB2, RAB5A, RAP2, RIG, S100A11, SCYA2, SEPP1,SGK, SKI, SLUG, TACC1, TASTIN, TBXA2F, TBXA2R, TFCP2, THROMBOSPONDIN1,TIMP2, TNFS10, TNFSF10, TOP2A, TRAF4, TRAP1, UBCH10, VAV2, VIMENTIN,VINCULIN, and YWHAB.
 12. The method of claim 1 wherein the marker is aprotein.
 13. The method of claim 1 wherein the marker is a messengerRNA.
 14. The method of claim 1 wherein the marker is a messenger RNA anddetecting expression of the marker comprises producing a complementaryDNA.
 15. The method of claim 1 wherein the amount of the marker isquantified.
 16. The method of claim 1 wherein detecting the MIPOL1-ETV1genetic rearrangement and the one or more markers is associated with thestage, aggressiveness, or progression of prostate cancer or the presenceor risk of metastasis in a subject that was the source of the biologicalsample.
 17. The method of claim 1 wherein the biological samplecomprises, or comprises a fraction of, a prostate biopsy sample,prostatectomy tissue sample, blood sample, urine sample, semen sample,prostatic secretion sample, plasma sample, serum sample, urinesupernatant, urine cell pellet, or prostate cell sample.
 18. The methodof claim 1 further comprising quantifying ETV1 expression.
 19. Themethod of claim 1 further comprising producing or receiving thebiological sample.
 20. The method of claim 1 further comprisingreporting the presence, absence, or amount of the MIPOL1-ETV1 geneticrearrangement and one or more of the markers.
 21. The method of claim 1further comprising providing an array comprising one or more probescomprising a sequence complementary to the MIPOL1-ETV1 geneticrearrangement and one or more probes comprising a sequence complementaryto the set of markers.
 22. The method of claim 6 further comprisingtreating the subject that was the source of the biological sample.
 23. Akit for detecting a MIPOL1-ETV1 genetic rearrangement and one or moremarkers associated with prostate cancer in a biological sample, the kitcomprising: a) a first component selected from the group consisting of:i) a probe directly labeled with a detectable label and comprising asequence that is complementary to a junction at which an ETV1 gene isinserted into a MIPOL1 gene; ii) a first probe directly labeled with adetectable label and comprising a sequence complementary to a MIPOL1gene and a second probe directly labeled with a detectable label andcomprising a sequence complementary to an ETV1 gene; or c) a firstamplification oligonucleotide comprising a sequence complementary to aMIPOL1 gene, a second amplification oligonucleotide comprising asequence complementary to an ETV1 gene, and a probe directly labeledwith a detectable label and comprising a sequence complementary to theproduct produced from the first and second primers; and b) a secondcomponent comprising a probe directly labeled with a detectable labeland for detecting a marker selected from the set of markers consistingof AMACR/P504S, PCA3, PCGEM1, prostein/P501S, P503S, P504S, P509S,P510S, prostase/P703P, P710P, prostate specific antigen (PSA), prostaticacid phosphatase (PAP), prostate binding protein (PBP), ABCC5(MDR5),ADAMTS1, AMACR, ANNEXINA11, ANNEXINA1, ANNEXINA4, APP, ARHB, ASNS, ATF2,C1S, C4BPA, C7, CATHEPSINB, CATHEPSINH, CAVEOLIN2, CCND2, CFLAR,CLUSTERIN, COL15A1, COL1A2, COL3A1, c-terminal binding protein, CTBP1,CTBP2, CYSTATINC, E2EPF, EDNRB, EGR1, EPHA1, ETS2, EZH2, FASN, FAT,FHL1, FIBRONECTIN1, FKBP5, FLS353, FOLH1, FOSB, FZD7, GELSOLIN, GP73,GSTM1, GSTM3, GSTM5, GSTP1, HEPSIN, HEVIN, IGFBP3, IGFBP5, IL1R1, IL1R2,ITGA1, ITGB4, ITM2C, JUN, KERATIN5, LIMK1, LUMICAN, MADH4, MAP3K10,MAPK6, MCAM, MEIS1, MEIS2, MMECD10, MOESIN, MPDZ, MTA1, MYBL2, MYLK,NBL1, NCK1, NRAS, PCM1, pim-1, PLA2G2A, PP1CB, PPP2CB, PRKCL2, PSG9,RAB2, RAB5A, RAP2, RIG, S100A11, SCYA2, SEPP1, SGK, SKI, SLUG, TACC1,TASTIN, TBXA2F, TBXA2R, TFCP2, THROMBOSPONDIN1, TIMP2, TNFS10, TNFSF10,TOP2A, TRAF4, TRAP1, UBCH10, VAV2, VIMENTIN, VINCULIN, and YWHAB. 24.The kit of claim 13 wherein the probe is a nucleic acid comprising asequence complementary to the marker.
 25. The kit of claim 13 whereinthe probe is an antibody specific for the marker.
 26. The kit of claim13 wherein the first component comprises a probe or a pair of probes andan array comprises the first and second components.
 27. An assaycomposition comprising a sample from a subject and: a) a first componentselected from the group consisting of: i) a probe directly labeled witha detectable label and comprising a sequence that is complementary to ajunction at which an ETV1 gene is inserted into a MIPOL1 gene; ii) afirst probe directly labeled with a detectable label and comprising asequence complementary to a MIPOL1 gene and a second probe directlylabeled with a detectable label and comprising a sequence complementaryto an ETV1 gene; or c) a first amplification oligonucleotide comprisinga sequence complementary to a MIPOL1 gene, a second amplificationoligonucleotide comprising a sequence complementary to an ETV1 gene, anda probe directly labeled with a detectable label and comprising asequence complementary to the product produced from the first and secondprimers; and b) a second component comprising a probe directly labeledwith a detectable label and for detecting a marker selected from the setof markers consisting of AMACR/P504S, PCA3, PCGEM1, prostein/P501S,P503S, P504S, P509S, P510S, prostase/P703P, P710P, prostate specificantigen (PSA), prostatic acid phosphatase (PAP), prostate bindingprotein (PBP), ABCC5(MDR5), ADAMTS1, AMACR, ANNEXINA11, ANNEXINA1,ANNEXINA4, APP, ARHB, ASNS, ATF2, C1S, C4BPA, C7, CATHEPSINB,CATHEPSINH, CAVEOLIN2, CCND2, CFLAR, CLUSTERIN, COL15A1, COL1A2, COL3A1,c-terminal binding protein, CTBP1, CTBP2, CYSTATINC, E2EPF, EDNRB, EGR1,EPHA1, ETS2, EZH2, FASN, FAT, FHL1, FIBRONECTIN1, FKBP5, FLS353, FOLH1,FOSB, FZD7, GELSOLIN, GP73, GSTM1, GSTM3, GSTM5, GSTP1, HEPSIN, HEVIN,IGFBP3, IGFBP5, IL1R1, IL1R2, ITGA1, ITGB4, ITM2C, JUN, KERATIN5, LIMK1,LUMICAN, MADH4, MAP3K10, MAPK6, MCAM, MEIS1, MEIS2, MMECD10, MOESIN,MPDZ, MTA1, MYBL2, MYLK, NBL1, NCK1, NRAS, PCM1, pim-1, PLA2G2A, PP1CB,PPP2CB, PRKCL2, PSG9, RAB2, RAB5A, RAP2, RIG, S100A11, SCYA2, SEPP1,SGK, SKI, SLUG, TACC1, TASTIN, TBXA2F, TBXA2R, TFCP2, THROMBOSPONDIN1,TIMP2, TNFS10, TNFSF10, TOP2A, TRAF4, TRAP1, UBCH10, VAV2, VIMENTIN,VINCULIN, and WTHAB.
 28. The assay composition of claim 17 wherein thesecond component comprises a probe that is a nucleic acid comprising asequence complementary to the marker.
 29. The assay composition of claim17 wherein the second component comprises an antibody that is specificfor the marker.
 30. The assay composition of claim 17 wherein a probe orprimer is hybridized to the MIPOL1-ETV1 genetic rearrangement and thesecond component is a nucleic acid hybridized to the marker.
 31. Theassay composition of claim 17 wherein a probe or primer is hybridized tothe MIPOL1-ETV1 genetic rearrangement and the second component is anantibody specifically bound to the marker.
 32. The assay composition ofclaim 17 wherein the first component comprises a first amplificationoligonucleotide comprising a sequence complementary to a MIPOL1 gene, asecond amplification oligonucleotide comprising a sequence complementaryto an ETV1 gene, a probe directly labeled with a detectable label andcomprising a sequence complementary to the product produced from thefirst and second primers, and a polymerase.